Combination of ABCG2 inhibitors with sacituzumab govitecan (IMMU-132) overcomes resistance to SN-38 in Trop-2 expressing cancers

ABSTRACT

The present invention relates to therapeutic ADCs comprising a drug attached to an anti-cancer antibody or antigen-binding antibody fragment. Preferably the drug is SN-38. More preferably the antibody or fragment thereof binds to Trop-2 and the therapy is used to treat a Trop-2 positive cancer. Most preferably the antibody is hRS7. The ADC is administered to a subject with a cancer in combination with an ABCG2 inhibitor. The combination therapy is effective to treat cancers that are resistant to drug alone and/or to ADC alone.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/429,671, filed Feb. 10, 2017, which claimed the benefit under 35U.S.C. 119(e) of U.S. Provisional Patent Application Nos. 62/293,530,filed Feb. 10, 2016, 62/329,788, filed Apr. 29, 2016, and 62/336,985,filed May 16, 2016, the text of each of which is incorporated herein byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 8, 2017, isnamed IMM366US1 SL.txt and is 21,436 bytes in size.

FIELD OF THE INVENTION

The present invention relates to use of combination therapy withanti-Trop-2 antibody drug conjugates (ADCs) and inhibitors of the ABC(ATP-binding cassette) transporters (preferably ABCG2), which areresponsible for drug resistance by promoting active drug efflux. Thecombination provides effective therapy against cancer cells and otherdisease-associated cells that are resistant to certain cytotoxic(chemotherapeutic) agents, such as camptothecins (e.g., SN-38,topotecan), anthracyclines (e.g., doxorubicin, daunorubicin),anthracenediones (e.g., mitoxantrone), taxanes (e.g., paclitaxel), vincaalkaloids (e.g., vincristine, vinblastine), epipodophyllotoxins (e.g.,etoposide, teniposide) and platinum compounds (e.g., cisplatinum).Combination therapy with ADCs and ABCG2 inhibitors provides effectivetreatment for cancers that are otherwise resistant to the conjugateddrug and/or ADC. In certain preferred embodiments, the conjugated drugmay be SN-38 and the ADC may be IMMU-132 (sacituzumab govitecan orhRS7-CL2A-SN-38). In other preferred embodiments, the ABCG2 inhibitormay be fumitremorgin C, Ko143, YHO-13351, or other known ABCG2inhibitors (e.g., curcumin, GF120918 (Elacridar), YHO-13177). Inalternative embodiments, inhibitors of other ABC transporters that areknown to be involved in drug resistance, such as ABCB1 or ABCC1, mayalso be used in combination therapy with one or more ADCs. Preferably,the combination therapy is effective to treat Trop-2 positive cancers inpatients who had relapsed from or shown resistance to treatmentscomprising irinotecan therapy, such as pancreatic cancer,triple-negative breast cancer, small cell lung cancer and non-small celllung cancer.

BACKGROUND OF THE INVENTION

Resistance of cancer cells to chemotherapy has been a major treatmentdifficulty for decades and results in approximately 90% of cancertreatment failures (Pluchino et al., 2012, Drug Resistance Updates 15:98-105). Certain cancers exhibit intrinsic resistance tochemotherapeutic agents, while in others drug resistance develops duringthe course of therapy (Lippert et al., 2011, Int J Med Sci 8: 245-253;Ricci et al., 2015, J Develop Drugs 4:138). Multiple drug resistance(MDR) is one of the most common ways in which cancer cells developresistance to chemotherapeutic agents, primarily due to the active drugefflux catalyzed by ATP binding cassette (ABC) transporters (Ricci etal., 2015, J Develop Drugs 4:138). While the ABC transporters comprise avery large superfamily with varied functions, only a few of thesetransporters appear to be involved in MDR, including ABCB1, ABCC1 andABCG2 (Ricci et al., 2015, J Develop Drugs 4:138). Of these, the ABCG2transporter is the predominant one expressed in solid tumors (Ricci etal., 2015, J Develop Drugs 4:138).

While chemotherapy with systemic drug administration has been widelyused in cancer treatment, more recently the targeted delivery of drugsin the form of antibody-drug conjugates has improved efficacy and safetyof anti-cancer agents. Such ADCs provide targeted delivery of theconjugated drug to cells expressing disease-associated antigens, such astumor associated antigens (TAAs), while reducing systemic exposure ofnormal tissues that have lower expression levels or do not express thetargeted antigen. Typically, the drug conjugate is releasedintracellularly after internalization of the ADC, although in some casesextracellular release from the cell-bound ADC may also occur. In eithercase, tumors may still exhibit intrinsic or developed resistant to theanti-cancer agent, due to the activity of ABC transporters. Thus,despite the notable success in treating diverse cancers,chemotherapeutics, including antibody-drug conjugates (ADCs), loseclinical activity over time, as exemplified by irinotecan (Xu et al,2002, Ann Oncol 13:1841-51), doxorubicin (Miller et al., 1988, J ClinOncol 6:880-8), paclitaxel (Orr et al., 2003, Oncogene 22:7280-95),cisplatin (Siddik, 2003, Oncogene 22:7265-79), gemtuzumab ozogamicin(Walter et al., 2007, Blood 109:4168-70), inotuzumab ozogamicin(Takeshita et al., 2009, Br J Haematol 146-34-43), and others (Szakacset al., 2006, Nat Rev Drug Discov 5:219-34).

The occurrence of drug resistance in cancer cells can result from avariety of factors (Housman et al., 2014, Cancers 6:1769-92), such asdecreased uptake of soluble drugs, activation of drug-detoxifyingsystems, modulation or mutation of drug targets, defective apoptosispathways, and above all, over-expression of one or more efflux pumps ofthe ATP-binding cassette (ABC) superfamily (Gottesman et al., 2002, NatRev Cancer 2:48-58). The human genome comprises a total of 49 genes inthe ABC superfamily (Vasiliou et al., 2009, Hum Genom 3:281-90), eachassigned to one of seven subfamilies (A through G) based on the orderand sequence homology of the transmembrane (TM) domain and thenucleotide-binding folds (NBFs). To date, ABCB1 (also known as MDR1 orP-gp), ABCC1 (also known as MRP1), and ABCG2 (also known as BCRP, MXR,or ABC-P), account for most studies on MDR (Ambudkar et al., 2003,Oncogene 22:7468-85; Cole, 2014, J Biol Chem 289:30880-8; Doyle andRoss, 2003, Oncogene 22:7340-58).

Members of the ABC superfamily are transmembrane proteins, expressedeither as a full- or half-transporter. A full-transporter typicallycontains two transmembrane (TM) domains and two nucleotide-binding folds(NBFs), with the TM domains participating in substrate recognition andtranslocation across the membrane, while the cytosolic NBFs provide thedriving force for transport via hydrolysis of the bound ATP. Bycontrast, a half-transporter has only one TM domain and one NBF, andmust form either homodimers or heterodimers to be functional. A notableexample of a full-transporter is ABCB1, whose substrates include vincaalkaloids, anthracyclines, epipodophyllotoxins, taxanes, irinotecan, andSN-38 (Szakacs et al., 2006, Nat Rev Drug Discov 5:219-34). The fivemembers of the ABCG subfamily are all half-transporters (Vasiliou etal., 2009, Hum Genom 3:281-90), of which ABCG2 has been identified forits role in mediating cellular resistance to SN-38 (Brangi et al., 1999,Cancer Res 59:5938-46; Kawabata et al., 2001, Biochem Biophys Res Commun280:1216-23), as well as to tyrosine kinase inhibitors (Ozvegy-Laczka etal., 2005, Drug Resist Updat 8:15-26).

With the molecular mechanisms of intrinsic and acquired drug resistancein cancer increasingly being delineated, multiple approaches tocircumvent MDR have emerged. For example, the sensitivity of inotuzumabozogamicin in ABCB1-expressing sublines of Daudi and Raji lymphomascould be restored effectively with PSC-833 (Walter et al., 2007, Blood109:4168-70), a second-generation modifier of ABCB1 (Twentyman andBleehan, 1991, Eur J Cancer 27:1639-42). The use of a hydrophilic linkerfor conjugating DM1 to antibodies also enabled such ADCs to evadeABCB1-mediated resistance (Kovton et al., 2010, Cancer Res 70:2528-37),presumably due to the generation of a cytotoxic metabolite that wasbetter retained by the ABCB1-expressing cells. In addition, targetingdetoxifying enzymes, such as glutathione S-transferase, withintracellularly activated prodrugs was found to be promising (Ramsay andDilda, 2014, Front Pharmacol 5:181). However, the rationale of usinginhibitors of ABC transporters to overcome MDR has met little success inclinical trials, which could be in part due to both imperfect inhibitorsand inadequate study design. This is being addressed by developing neweragents with greater substrate specificity, higher potency, lowertoxicity, and improved pharmacokinetic properties.

Sacituzumab govitecan, hereafter referred to as IMMU-132, is aTrop-2-targeting ADC of SN-38, the active metabolite of irinotecan.IMMU-132 departs from most ADCs in its use of a moderately, notultratoxic drug, its high drug to antibody ratio (DAR) without impairingtarget affinity and pharmacokinetics, and its selection of apH-sensitive, cleavable linker to confer cytotoxicity to both tumor andbystander cells (Cardillo et al., 2011, Clin Cancer Res 17:3157-69;Cardillo et al., 2015, Bioconjugate Chem 26:919-31; Goldenberg et al.,2015, Oncotarget 6:22496-512). This novel ADC is currently in clinicaltrials for patients with advanced triple-negative breast cancer(Starodub et al., 2015, Clin Cancer Res 21:3870-8), urothelial bladdercancer (Faltas et al., 2016, Clin Genitourinary Cancer 14:e75-9) andother solid cancers. Since these patients were all heavily pretreatedwith chemotherapy, the presence of acquired resistance with theexpression of MDR genes is highly likely, which may affect thetherapeutic outcome of IMMU-132. A need exists for improved methods ofuse of ADCs, such as IMMU-132, to treat tumors that are resistant tochemotherapy.

SUMMARY OF THE INVENTION

The present invention resolves an unfulfilled need in the art byproviding improved methods and compositions for treating drug-resistanttumors with a combination of anti-Trop-2 ADC and an inhibitor of ABCtransporters, preferably an inhibitor of ABCG2. More preferably, the ADCis conjugated to SN-38. However, the person of ordinary skill willrealize that alternative drugs may be incorporated in the ADC. Thedisclosed methods and compositions are of use for the treatment of avariety of diseases and conditions which are refractory or lessresponsive to other forms of therapy. Preferred diseases or conditionsthat may be treated with the subject combination therapy include, forexample, Trop-2 positive cancer, such as metastatic pancreatic cancer,triple-negative breast cancer, urothelial cancer, small cell lung canceror non-small cell lung cancer.

Preferably, the ADC incorporates an anti-Trop-2 antibody, antibodyfragment, bispecific or other multivalent antibody, or otherantibody-based molecule or compound. The antibody can be of variousisotypes, preferably human IgG1, IgG2, IgG3 or IgG4, more preferablycomprising human IgG1 hinge and constant region sequences. The antibodyor fragment thereof can be a chimeric human-mouse, a chimerichuman-primate, a humanized (human framework and murine hypervariable(CDR) regions), or fully human antibody, as well as variations thereof,such as half-IgG4 antibodies (referred to as “unibodies”), as describedby van der Neut Kolfschoten et al. (Science 2007; 317:1554-1557). Morepreferably, the antibody or fragment thereof may be designed or selectedto comprise human constant region sequences that belong to specificallotypes, which may result in reduced immunogenicity when the ADC isadministered to a human subject. Preferred allotypes for administrationinclude a non-G1m1 allotype (nG1m1), such as G1m3, G1m3,1, G1m3,2 orG1m3,1,2. More preferably, the allotype is selected from the groupconsisting of the nG1m1, G1m3, nG1m1,2 and Km3 allotypes.

In alternative embodiments, the combination of anti-Trop-2 ADC and ABCG2inhibitor may be administered alone, or else in further combination withanother anti-cancer antibody. Antibodies against a wide variety of humantumor-associated antigens (TAAs) are known. Such TAAs, include but notlimited to, carbonic anhydrase IX, alpha-fetoprotein (AFP), α-actinin-4,A3, antigen specific for A33 antibody, ART-4, B7, Ba 733, BAGE,BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCL19, CCL21, CD1, CD1a,CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20,CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40,CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67,CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138,CD147, CD154, CDC27, CDK-4/m, CDKN2A, CTLA-4, CXCR4, CXCR7, CXCL12,HIF-1α, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, c-Met,DAM, EGFR, EGFRvIII, EGP-1 (Trop-2), EGP-2, ELF2-M, Ep-CAM, fibroblastgrowth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE,gp100, GRO-β, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and itssubunits, HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M,HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R,IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18,IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KC4-antigen,KS-1-antigen, KS1-4, Le-Y, LDR/FUT, macrophage migration inhibitoryfactor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP,MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13,MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, PAM4 antigen, pancreaticcancer mucin, PD-1 receptor, placental growth factor, p53, PLAGL2,prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, ILGF, ILGF-1R, IL-6,IL-25, RS5, RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC,TAG-72, tenascin, TRAIL receptors, TNF-α, Tn antigen,Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-Bfibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a,C5, an angiogenesis marker, bcl-2, bcl-6, Kras, an oncogene marker andan oncogene product (see, e.g., Sensi et al., Clin Cancer Res 2006,12:5023-32; Parmiani et al., J Immunol 2007, 178:1975-79; Novellino etal. Cancer Immunol Immunother 2005, 54:187-207). Preferably, theantibody binds to CEACAM5, CEACAM6, Trop-2, MUC-16, AFP, MUC5ac, CD74,CD19, CD20, CD22 or HLA-DR.

Exemplary antibodies that may be utilized include, but are not limitedto, hR1 (anti-IGF-1R, U.S. patent application Ser. No. 12/722,645, filedMar. 12, 2010), hPAM4 (anti-mucin, U.S. Pat. No. 7,282,567), hA20(anti-CD20, U.S. Pat. No. 7,251,164), hA19 (anti-CD19, U.S. Pat. No.7,109,304), hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1(anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22, U.S. Pat. No.7,074,403), hMu-9 (anti-CSAp, U.S. Pat. No. 7,387,773), hL243(anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM5, U.S. Pat.No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Pat. No. 7,541,440), hRS7(anti-EGP-1, U.S. Pat. No. 7,238,785), hMN-3 (anti-CEACAM6, U.S. Pat.No. 7,541,440), the Examples section of each cited patent or applicationincorporated herein by reference. More preferably, the antibody isIMMU-31 (anti-AFP), hRS7 (anti-Trop-2), hMN-14 (anti-CEACAM5), hMN-3(anti-CEACAM6), hMN-15 (anti-CEACAM6), hLL1 (anti-CD74), hLL2(anti-CD22), hL243 or IMMU-114 (anti-HLA-DR), hA19 (anti-CD19) or hA20(anti-CD20). As used herein, the terms epratuzumab and hLL2 areinterchangeable, as are the terms veltuzumab and hA20, hL243g4P,hL243gamma4P and IMMU-114.

Alternative antibodies of use include, but are not limited to, abciximab(anti-glycoprotein IIb/IIIa), alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab(anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab(anti-CD20), trastuzumab (anti-ErbB2), lambrolizumab (anti-PD-1receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4),abagovomab (anti-CA-125), adecatumumab (anti-EpCAM), atlizumab(anti-IL-6 receptor), benralizumab (anti-CD125), obinutuzumab (GA101,anti-CD20), CC49 (anti-TAG-72), AB-PGI-XG1-026 (anti-PSMA, U.S. patentapplication Ser. No. 11/983,372, deposited as ATCC PTA-4405 andPTA-4406), D2/B (anti-PSMA, WO 2009/130575), tocilizumab (anti-IL-6receptor), basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab(anti-CD11a), GA101 (anti-CD20; Glycart Roche), muromonab-CD3 (anti-CD3receptor), natalizumab (anti-a4 integrin), omalizumab (anti-IgE);anti-TNF-α antibodies such as CDP571 (Ofei et al., 2011, Diabetes45:881-85), MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, M303 (ThermoScientific, Rockford, Ill.), infliximab (Centocor, Malvern, Pa.),certolizumab pegol (UCB, Brussels, Belgium), anti-CD40L (UCB, Brussels,Belgium), adalimumab (Abbott, Abbott Park, Ill.), Benlysta (Human GenomeSciences); antibodies for therapy of Alzheimer's disease such as Alz 50(Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47), gantenerumab,solanezumab and infliximab; anti-fibrin antibodies like 59D8, T2G1s,MH1; anti-CD38 antibodies such as MOR03087 (MorphoSys AG), MOR202(Celgene), HuMax-CD38 (Genmab) or daratumumab (Johnson & Johnson).

In a preferred embodiment, the chemotherapeutic moiety is selected fromcamptothecin (CPT) and its analogs and derivatives and is morepreferably SN-38. However, other chemotherapeutic moieties that may beutilized include taxanes (e.g, baccatin III, taxol), epothilones,anthracyclines (e.g., doxorubicin (DOX), epirubicin,morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin(cyanomorpholino-DOX), 2-pyrrolinodoxorubicin (2-PDOX) or a prodrug formof 2-PDOX (pro-2-PDOX); see, e.g., Priebe W (ed.), ACS symposium series574, published by American Chemical Society, Washington D.C., 1995(332pp) and Nagy et al., Proc. Natl. Acad. Sci. USA 93:2464-2469, 1996),benzoquinoid ansamycins exemplified by geldanamycin (DeBoer et al.,Journal of Antibiotics 23:442-447, 1970; Neckers et al., Invest. NewDrugs 17:361-373, 1999), and the like. Preferably, the antibody orfragment thereof links to at least one chemotherapeutic moiety;preferably 1 to about 5 chemotherapeutic moieties; more preferably 6 ormore chemotherapeutic moieties, most preferably about 6 to about 8chemotherapeutic moieties.

Various embodiments may concern use of the subject methods andcompositions to treat a cancer, including but not limited tonon-Hodgkin's lymphomas, B-cell acute and chronic lymphoid leukemias,Burkitt lymphoma, Hodgkin's lymphoma, acute large B-cell lymphoma, hairycell leukemia, acute myeloid leukemia, chronic myeloid leukemia, acutelymphocytic leukemia, chronic lymphocytic leukemia, T-cell lymphomas andleukemias, multiple myeloma, Waldenstrom's macroglobulinemia,carcinomas, melanomas, sarcomas, gliomas, bone, and skin cancers. Thecarcinomas may include carcinomas of the oral cavity, esophagus,gastrointestinal tract, pulmonary tract, lung, stomach, colon, breast,ovary, prostate, uterus, endometrium, cervix, urinary bladder,urothelium, pancreas, bone, brain, connective tissue, liver, gallbladder, urinary bladder, kidney, skin, central nervous system andtestes. Preferably, the cancer expresses Trop-2 antigen.

In certain embodiments involving treatment of cancer, therapy withanti-Trop-2 ADC and ABCG2 inhibitor may be used in combination withstandard anti-cancer treatments, such as surgery, radiation therapy,chemotherapy, immunotherapy with naked antibodies, radioimmunotherapy,immunomodulators, vaccines, and the like. These combination therapiescan allow lower doses of each therapeutic to be given in suchcombinations, thus reducing certain severe side effects, and potentiallyreducing the courses of therapy required. When there is no or minimaloverlapping toxicity, ful doses of each can also be given.

Preferred optimal dosing of ADC may include a dosage of between 3 mg/kgand 18 mg/kg, preferably given either weekly, twice weekly or everyother week. The optimal dosing schedule may include treatment cycles oftwo consecutive weeks of therapy followed by one, two, three or fourweeks of rest, or alternating weeks of therapy and rest, or one week oftherapy followed by two, three or four weeks of rest, or three weeks oftherapy followed by one, two, three or four weeks of rest, or four weeksof therapy followed by one, two, three or four weeks of rest, or fiveweeks of therapy followed by one, two, three, four or five weeks ofrest, or administration once every two weeks, once every three weeks oronce a month. Treatment may be extended for any number of cycles,preferably at least 2, at least 4, at least 6, at least 8, at least 10,at least 12, at least 14, or at least 16 cycles. The dosage may be up to24 mg/kg. Exemplary dosages of use may include 1 mg/kg, 2 mg/kg, 3mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg,11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg,and 18 mg/kg. Preferred dosages are 4, 6, 8, 9, 10, or 12 mg/kg. Theperson of ordinary skill will realize that a variety of factors, such asage, general health, specific organ function or weight, as well aseffects of prior therapy on specific organ systems (e.g., bone marrow)may be considered in selecting an optimal dosage of ADC, and that thedosage and/or frequency of administration may be increased or decreasedduring the course of therapy. The dosage may be repeated as needed, withevidence of tumor shrinkage observed after as few as 4 to 8 doses. Theoptimized dosages and schedules of administration disclosed herein showunexpected superior efficacy and reduced toxicity in human subjects.Surprisingly, the superior efficacy allows treatment of tumors that werepreviously found to be resistant to one or more standard anti-cancertherapies, including the parental compound, CPT-11 (irinotecan), fromwhich SN-38 is derived in vivo.

The subject methods may include use of CT and/or PET/CT, or MRI, tomeasure tumor response at regular intervals. Blood levels of tumormarkers, such as CEA (carcinoembryonic antigen), CA19-9, AFP, CA 15.3,or PSA, may also be monitored. Dosages and/or administration schedulesmay be adjusted as needed, according to the results of imaging and/ormarker blood levels.

A surprising result with the instant claimed compositions and methods isthe unexpected tolerability of high doses of antibody-drug conjugate,even with repeated infusions, with only relatively low-grade toxicitiesof nausea and vomiting observed, or manageable neutropenia. A furthersurprising result is the lack of accumulation of the antibody-drugconjugate, unlike other products that have conjugated SN-38 to albumin,PEG or other carriers. The lack of accumulation is associated withimproved tolerability and lack of serious toxicity even after repeatedor increased dosing. These surprising results allow optimization ofdosage and delivery schedule, with unexpectedly high efficacies and lowtoxicities. The claimed methods provide for shrinkage of solid tumors,in individuals with previously resistant cancers, of 15% or more,preferably 20% or more, preferably 30% or more, more preferably 40% ormore in size (as measured by longest diameter). The person of ordinaryskill will realize that tumor size may be measured by a variety ofdifferent techniques, such as total tumor volume, maximal tumor size inany dimension or a combination of size measurements in severaldimensions. This may be with standard radiological procedures, such ascomputed tomography, ultrasonography, and/or positron-emissiontomography. The means of measuring size is less important than observinga trend of decreasing tumor size with ADC and ABCG2 inhibitor treatment,preferably resulting in elimination of the tumor.

While the ADC may be administered as a periodic bolus injection, inalternative embodiments the ADC may be administered by continuousinfusion of antibody-drug conjugate. In order to increase the Cmax andextend the PK of the ADC in the blood, a continuous infusion may beadministered for example by indwelling catheter. Such devices are knownin the art, such as HICKMAN®, BROVIAC® or PORT-A-CATH® catheters (see,e.g., Skolnik et al., Ther Drug Monit 32:741-48, 2010) and any suchknown indwelling catheter may be used. A variety of continuous infusionpumps are also known in the art and any such known infusion pump may beused. The dosage range for continuous infusion may be between 0.1 and3.0 mg/kg per day. More preferably, these ADCs can be administered byintravenous infusions over relatively short periods of 2 to 5 hours,more preferably 2-3 hours.

In particularly preferred embodiments, the ADC and dosing schedules maybe efficacious in patients resistant to standard therapies. For example,an anti-Trop-2 hRS7-SN-38 ADC, in combination with ABCG2 inhibitor, maybe administered to a patient who has not responded to prior therapy withirinotecan, the parent agent of SN-38. Surprisingly, theirinotecan-resistant patient may show a partial or even a completeresponse to hRS7-SN-38 and ABCG2 inhibitor. The ability of the ADC tospecifically target the tumor tissue may overcome tumor resistance byimproved targeting and enhanced delivery of the therapeutic agent, whilethe combination of ADC and ABCG2 inhibitor may be effective in tumorswith active transport mechanisms for eliminating SN-38. Otherantibody-SN-38 ADCs, such as IMMU-130 (anti-CEACAM-5-SN-38) or IMMU-140(anti-HLA-DR-SN-38) may show similar improved efficacy and/or decreasedtoxicity when used with an ABCG2 inhibitor, compared to alternativestandard therapeutic treatments. A specific preferred subject may be ametastatic colorectal cancer patient, metastatic pancreatic cancerpatient, a triple-negative breast cancer patient, a HER+, ER+,progesterone+breast cancer patient, a metastatic non-small-cell lungcancer (NSCLC) patient, a metastatic small-cell lung cancer patient, ametastatic stomach cancer patient, a metastatic renal cancer patient, ametastatic urinary bladder cancer patient, a metastatic ovarian cancerpatient, a metastatic urothelial cancer patient, or a metastatic uterinecancer patient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Preclinical in vivo therapy of athymic nude mice, bearing Capan1 human pancreatic carcinoma, with SN-38 conjugates of hRS7(anti-Trop-2), hPAM4 (anti-MUC5ac), hMN-14 (anti-CEACAM5) ornon-specific control hA20 (anti-CD20).

FIG. 2. Preclinical in vivo therapy of athymic nude mice, bearing BxPC3human pancreatic carcinoma, with anti-TROP2-CL2A-SN-38 conjugatescompared to controls.

FIG. 3. ADCC of various hRS7-ADCs vs. hRS7 IgG.

FIG. 4A. Structures of CL2-SN-38 and CL2A-SN-38.

FIG. 4B. Comparative efficacy of anti-Trop-2 ADC linked to CL2 vs. CL2Alinkers versus hA20 ADC and saline control, using COLO 205 colonicadenocarcinoma. Animals were treated twice weekly for 4 weeks asindicated by the arrows. COLO 205 mice (N=6) were treated with 0.4 mg/kgADC and tumors measured twice a week.

FIG. 4C. Comparative efficacy of anti-Trop-2 ADC linked to CL2 vs. CL2Alinkers versus hA20 ADC and saline control, using Capan-1 pancreaticadenocarcinoma. Animals were treated twice weekly for 4 weeks asindicated by the arrows. Capan-1 mice (N=10) were treated with 0.2 mg/kgADC and tumors measured weekly.

FIG. 5A. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). Mice bearing Calu-3tumors (N=5-7) were injected with hRS7-CL2-SN-38 every 4 days for atotal of 4 injections (q4dx4).

FIG. 5B. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). COLO 205 tumor-bearingmice (N=5) were injected 8 times (q4dx8) with the ADC or every 2 daysfor a total of 5 injections (q2dx5) with the MTD of irinotecan.

FIG. 5C. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). Capan-1 (N=10) weretreated twice weekly for 4 weeks with the agents indicated.

FIG. 5D. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). BxPC-3 tumor-bearingmice (N=10) were treated twice weekly for 4 weeks with the agentsindicated.

FIG. 5E. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). In addition to ADCgiven twice weekly for 4 week, SK-MES-1 tumor-bearing (N=8) micereceived the MTD of CPT-11 (q2dx5).

FIG. 6A. Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster mice.Fifty-six Swiss-Webster mice were administered 2 i.p. doses of buffer orthe hRS7-CL2A-SN-38 3 days apart (4, 8, or 12 mg/kg of SN-38 per dose;250, 500, or 750 mg conjugate protein/kg per dose). Seven and 15 daysafter the last injection, 7 mice from each group were euthanized, withblood counts and serum chemistries performed. Graphs show the percent ofanimals in each group that had elevated levels of AST.

FIG. 6B. Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster mice.Fifty-six Swiss-Webster mice were administered 2 i.p. doses of buffer orthe hRS7-CL2A-SN-38 3 days apart (4, 8, or 12 mg/kg of SN-38 per dose;250, 500, or 750 mg conjugate protein/kg per dose). Seven and 15 daysafter the last injection, 7 mice from each group were euthanized, withblood counts and serum chemistries performed. Graphs show the percent ofanimals in each group that had elevated levels of ALT.

FIG. 6C. Tolerability of hRS7-CL2A-SN-38 in Cynomolgus monkeys. Sixmonkeys per group were injected twice 3 days apart with buffer (control)or hRS7-CL2A-SN-38 at 0.96 mg/kg or 1.92 mg/kg of SN-38 equivalents perdose (60 and 120 mg/kg conjugate protein). All animals were bled on day−1, 3, and 6. Four monkeys were bled on day 11 in the 0.96 mg/kg group,3 in the 1.92 mg/kg group. Changes in neutrophil counts in Cynomolgusmonkeys.

FIG. 6D. Tolerability of hRS7-CL2A-SN-38 in Cynomolgus monkeys. Sixmonkeys per group were injected twice 3 days apart with buffer (control)or hRS7-CL2A-SN-38 at 0.96 mg/kg or 1.92 mg/kg of SN-38 equivalents perdose (60 and 120 mg/kg conjugate protein). All animals were bled on day−1, 3, and 6. Four monkeys were bled on day 11 in the 0.96 mg/kg group,3 in the 1.92 mg/kg group. Changes in platelet counts in Cynomolgusmonkeys.

FIG. 7A. Comparison of in vitro efficacy of anti-Trop-2 ADCs (hRS7-SN-38versus MAB650-SN-38) in Capan-1 human pancreatic adenocarcinoma.

FIG. 7B. Comparison of in vitro efficacy of anti-Trop-2 ADCs (hRS7-SN-38versus MAB650-SN-38) in BxPC-3 human pancreatic adenocarcinoma.

FIG. 7C. Comparison of in vitro efficacy of anti-Trop-2 ADCs (hRS7-SN-38versus MAB650-SN-38) in NCI-N87 human gastric adenocarcinoma.

FIG. 8A. Comparison of in vitro efficacy of 162-46.2-SN-38 vs.hRS7-SN-38 in BxPC-3 human pancreatic adenocarcinoma cells.

FIG. 8B. Comparison of in vitro efficacy of 162-46.2-SN-38 vs.hRS7-SN-38 in MDA-MB-468 human breast adenocarcinoma cells.

FIG. 9. IMMU-132 phase I/II data for best response by RECIST criteria.

FIG. 10. IMMU-132 phase I/II data for time to progression and bestresponse (RECIST).

FIG. 11. Therapeutic efficacy of murine anti-Trop-2-SN-38 ADC(162-46.2-SN-38) compared to hRS7-SN-38 in mice bearing NCI-N87 humangastric carcinoma xenografts.

FIG. 12. Accumulation of SN-38 in tumors of nude mice with Capan-1 humanpancreatic cancer xenografts, when administered as free irinotecan vs.IMMU-132 ADC.

FIG. 13. Individual patient demographics and prior treatment for phaseI/II IMMU-132 anti-Trop-2 ADC in pancreatic cancer patients.

FIG. 14. Response assessment to IMMU-132 anti-Trop-2 ADC in pancreaticcancer patients.

FIG. 15. Summary of time to progression (TTP) results in humanpancreatic cancer patients administered IMMU-132 anti-Trop-2 ADC.

FIG. 16. Adverse events observed in phase I study of IMMU-132 in varioustumor types.

FIG. 17. Response assessment in sacituzumab govitecan-treated patients.(A) Composite schematic showing the best response (y-axis) determinedfrom target lesion measurements according to RECIST 1.1 and thetime-to-progression (z-axis; TTP expressed in months), measured from thedate of the first dose until CT documentation of progression as perRECIST. Best response bars are color-coded to identify the 4 startingdose levels. Four of the 25 patients (numbers 6, 9, 14, and 23; 2 PDC, 1GC and 1 SCLC) who were classified with disease progression are notshown because either they did not have a follow-up CT with measurementof target lesions or they had new lesions despite having stable targetlesions measurements. A bar break (//) shown for two PD patients denotestarget lesions increased >30%, whereas TTP values in the boxes at top ofthe graph show the patients who exceeded 9 months. The number of priortherapies (in parentheses) and the patients who received priortopoisomerase I therapy (asterisks) are indicated below the graph. (B)Graph showing the patients sorted according to survival, showing alsotheir TTP. Survival data were unavailable for 2 PDC patients (numbers 6and 17 with TTP 1.0 and 2.9 months).

FIG. 18. CT response assessment in 2 of 3 patients with >30% reductionin target lesions. Patient 22 is a 65-year-old woman with poorlydifferentiated SCLC (Trop-2 expression by immunohistology, 3+) who hadreceived 2 months of carboplatin/etoposide (topoisomerase-II inhibitor)and 1 month of topotecan (topoisomerase-I inhibitor) with no response,followed by local radiation for 6 weeks (3000 cGy), but progressed. Fourweeks later, the patient started sacituzumab govitecan at 12 mg/kg (2doses), which was reduced to 9.0 mg/kg (1 dose), and finally to 6.75mg/kg for 9 doses. The patient presented initially with the sum of thelongest diameters (SLD) of the target lesions totaling 19.3 cm. Two ofthe target lesions showing the best shrinkage are shown at baseline (Aand C). After 4 treatments, she had a 38% reduction in target lesions,including a substantial reduction in the main lung lesion (5.8 to 2.7cm; panels B and D). On her next CT assessment 12 weeks later, thepatient progressed. Patient 3 a 62 year-old woman, who 5 months afterher initial diagnosis and surgery for colon cancer had a hepaticresection for liver metastases and then received 7 months of treatmentwith FOLFOX and 1 month of only 5-fluorouracil. She was referred to thesacituzumab govitecan trial with multiple lesions, primarily in theliver (left panels A, C, and E). Immunohistology showed a 2+ staining ofher primary cancer; her plasma CEA was 781 ng/mL. Therapy was initiatedat 8 mg/kg and 6 treatments later (12 weeks), the 3 target lesions hadreduced from 7.9 cm to 5.0 cm (−37%; PR). The response was confirmed 6.6weeks later (after ten doses), with additional shrinkage to 3.8 cm(−52%). Panels B, D, and F show the shrinkage of these 3 lesions (59%reduction at this time) 32 weeks from the start of treatment and afterreceiving 18 doses The patient continued therapy, achieving a maximumtumor reduction of 65% 10 months after treatment was initiated (28doses). Plasma CEA decreased to 26.5 ng/mL after 18 doses, butthereafter began to increase despite continued radiological evidence(target and non-target lesions) of additional disease reduction orstabilization. Approximately 1 year from the start of treatment (31doses given), one of the 3 target lesions progressed.

FIG. 19. Therapeutic efficacy of IMMU-132 with different DARs. NCI-N87human gastric carcinoma xenografts (subcutaneous) were established asdescribed in Methods. (A) Four groups of mice (N=9) were injected IVwith 2×0.5 mg (arrows) of IMMU-132 conjugates prepared with a DAR=6.89,3.28, or 1.64. Control animals received saline. Therapy began 7 daysafter tumor cells were administered (size was 0.248±0.047 cm³). Survivalcurves were generated based on the time to progression to >1.0 cm³, andwere analyzed by log-rank test (significance at P<0.05). (B) NCI-N87tumor-bearing mice (N=7-9; starting size=0.271±0.053 cm³) were treatedwith either 0.5 mg IMMU-132 (DAR=6.89) or 1.0 mg DAR=3.28 twice weeklyfor two weeks (arrows). Mice were euthanized and deemed to havesuccumbed to disease once tumors grew to >1.0 cm³. Profiles ofindividual tumor growth were obtained through linear-curve modeling.Statistical analysis of tumor growth was based on area under the curve(AUC) performed up to the time that the first animal within a group waseuthanized due to disease progression. An f-test was employed todetermine equality of variance between groups prior to statisticalanalysis of growth curves. A two-tailed t-test was used to assessstatistical significance between the various treatment groups andcontrols, except for the saline control, where a one-tailed t-test wasused (significance at P<0.05).

FIG. 20. Therapeutic efficacy of IMMU-132 in TNBC xenograft models. (A)Twenty-two days after s.c. implantation of MDA-MB-468 tumors (at theonset of treatment, tumors averaged 0.223±0.055 cm³), nude mice (7-8 pergroup) were injected IV with IMMU-132 or a control hA20 anti-CD20-SN-38conjugate twice weekly for two weeks (0.12 or 0.20 mg/kg SN-38equivalents per dose). Other animals were given irinotecan (10mg/kg/dose; SN-38 equivalent based on mass=5.8 mg/kg) IV every other dayfor 10 days for a total of five injections. (B) Starting on day 56 aftertreatment initiation, all animals in the control hA20-SN-38 group weregiven IMMU-132 (4×0.2 mg/kg SN-38 equivalents). The size of the tumorsin the individual animals of this group from the onset of tumortransplantation is given. Red arrows indicate when the hA20-SN-38conjugate was first given, and purple arrows indicate when the treatmentwith IMMU-132 was initiated. (C) Mice (N=12) bearing the MDA-MB-231 TNBCcell line (0.335±0.078 cm³) were treated with IMMU-132 or the controlhA20-SN-38 conjugate (0.4 mg/kg SN-38 equivalents), irinotecan (6.5mg/kg; ˜3.8 mg/kg SN-38 equivalents), or a combination of hRS7 IgG (25mg/kg) plus irinotecan (6.5 mg/kg).

FIG. 21. ADCC activity of IMMU-132. Specific cell lysis of target cellsby human PBMCs mediated by IMMU-132 was compared to parental hRS7.Target cells were plated the night before and the assay performed asdescribed in the Examples below. (A) MDA-MB-468 target cells. (B)NIH:OVCAR-3 target cells. (C) BxPC-3 target cells. *hRS7 versus all theother test agents (P<0.0054). **IMMU-132 versus negative controlshLL2-SN-38 and hLL2 (P<0.0003). *** IMMU-132 versus negative controlhLL2-CL2ASN-38 (P<0.0019).

FIG. 22. Pharmacokinetics of IMMU-132 in mice. Naive nude mice (N=5)were injected i.v. with 200 μg of IMMU-132. At various time-points thesemice were bled and serum obtained and analyzed for intact conjugate andcarrier hRS7 antibody, as described in the Examples below. Forcomparison, another group of mice was injected with 200 μg parentalhRS7. (A) Serum concentration and clearance of hRS7 from parentalcontrol injected mice. Concentration and clearance of (B) hRS7 carrierantibody versus (C) intact conjugate from IMMU-132 injected mice.Graphed data shown as mean±S.D.

FIG. 23. Efficacy of IMMU-132 in mice bearing human gastric carcinomaxenograft. Mice bearing NCI-N87 human gastric tumors (TV=0.249±0.049cm³) were treated with 0.35 mg IMMU-132 twice weekly for four weeks. (A)Mean tumor growth curves for IMMU-132 treated animals compared to salineand non-tumor-targeting control ADC, hA20-CL2A-SN-38, treated mice.Arrows indicate therapy days. (B) Survival curves for treated mice witha disease end-point of tumor progression greater than 1.0 cm³.

FIG. 24. Various IMMU-132 dosing schemes in mice bearing pancreatic andgastric tumor xenografts. Nude mice (N=8-10) bearing s.c. BxPC-3 orNCI-N87 xenografts were prepared. (A) BxPC-3-bearing mice were treated(arrows) with two cycles of IMMU-132 at 1 mg every 14 d, 0.5 mg weeklyfor two weeks, or 0.25 mg twice weekly for two weeks, totaling 2 mgIMMU-132 to all the mice. (B) Similar dosing of NCIN87-bearing mice(arrows), with mice in the 1-mg treatment group receiving one additionalcycle. (C) Chronic dosing of IMMU-132 in mice bearing NCI-N87, using0.5-mg once weekly for 2 weeks in a 3-week treatment cycle for a totalof 4 cycles. Corresponding survival curves (end-point: tumorprogression >1.0 cm3) to right of each tumor growth curve.

FIG. 25. Responses in 52 human TNBC patients treated with 10 mg/kgIMMU-132, after failing numerous prior therapies.

FIG. 26. Time to progression for CR+PR+SD in TNBC patients treated with10 mg/kg IMMU-132.

FIG. 27. Progression-free survival in TNBC patients treated with 10mg/kg IMMU-132.

FIG. 28. Best response in 29 assessable human NSCLC patients treatedwith 8 to 10 mg/kg IMMU-132.

FIG. 29. Time to progression in NSCLC patients treated with 8-10 mg/kgIMMU-132.

FIG. 30. Progression-free survival in NSCLC patients treated with 8 or10 mg/kg IMMU-132.

FIG. 31. Best response in 25 assessable human SCLC patients treated with8 to 10 mg/kg IMMU-132.

FIG. 32. Time to progression in SCLC patients treated with 8-10 mg/kgIMMU-132.

FIG. 33. Progression-free survival in SCLC patients treated with 8 or 10mg/kg IMMU-132.

FIG. 34. Best response in 11 assessable human urothelial cancer patientstreated with IMMU-132.

FIG. 35. Time to progression in urothelial cancer patients treated withIMMU-132.

FIG. 36. γH2AX assay for DSB by flow cytometry. Cells were treated ornot treated with SN-38 (250 nM) for 3 h and the levels of γH2AX weremonitored hourly by flow cytometry and shown as MFI in bar diagram (A)or as percent of untreated in (B). The effect of FTC (10 μM) to increasethe formation of DSB/γH2AX was shown for MDA-MB-231-5120 treated withSN-38 (C) and for NCI-N87-S120 treated for either SN-38 or IMMU-132 (D).

FIG. 37. Dose response curves of parental and S-120 cells treated withdifferent concentrations of SN-38 in the absence and presence ofYHO-13351 or Ko143. Reversal of SN-38-resistance by YHO-13351 was shownfor MDA-MB-231-S120 in (A), NCI-N87-S120 in (B), and by Ko143 forNCI-N87-S120 in (C).

FIG. 38. Efficacy of IMMU-132 in mice bearing SN-38-resistantNCI-N87-S-120 gastric carcinoma xenograft. (A) Mean tumor growth curvesfor NCI-N87 and NCI-N87-S-120 xenografts. (B) Mice bearing NCI-N87-S-120SN-38-resistant human gastric tumors were treated with IMMU-132,irinotecan, YHO-13551, or combinations as indicated on the graph anddescribed in Materials and Methods. (C) Mice bearing parental NCI-N87tumors treated with IMMU-132 or irinotecan at the same dose and scheduleas used in NCI-N87-S-120 tumor-bearing animals. In the survival curvesof (B) and (C), the starting day of therapy (when tumor volumes reachedapproximately 0.25 cm³) was marked as Day 0. Mice were euthanized oncetumors grew to >1.0 cm³ in size.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of theclaimed subject matter. Terms that are not expressly defined herein areused in accordance with their plain and ordinary meanings.

Unless otherwise specified, a or an means “one or more.”

The term about is used herein to mean plus or minus ten percent (10%) ofa value. For example, “about 100” refers to any number between 90 and110.

An antibody, as used herein, refers to a full-length (i.e., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an antigen-binding portion of an immunoglobulin molecule,such as an antibody fragment. An antibody or antibody fragment may beconjugated or otherwise derivatized within the scope of the claimedsubject matter. Such antibodies include but are not limited to IgG1,IgG2, IgG3, IgG4 (and IgG4 subforms), as well as IgA isotypes. As usedbelow, the abbreviation “MAb” may be used interchangeably to refer to anantibody, antibody fragment, monoclonal antibody or multispecificantibody.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv (single chain Fv), single domain antibodies(DABs or VHHs) and the like, including the half-molecules of IgG4 citedabove (van der Neut Kolfschoten et al. (Science 2007; 317 (14September):1554-1557). Regardless of structure, an antibody fragment ofuse binds with the same antigen that is recognized by the intactantibody. The term “antibody fragment” also includes synthetic orgenetically engineered proteins that act like an antibody by binding toa specific antigen to form a complex. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains and recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”). The fragments may be constructed in different ways toyield multivalent and/or multispecific binding forms.

A naked antibody is generally an entire antibody that is not conjugatedto a therapeutic agent. A naked antibody may exhibit therapeutic and/orcytotoxic effects, for example by Fc-dependent functions, such ascomplement fixation (CDC) and ADCC (antibody-dependent cellcytotoxicity). However, other mechanisms, such as apoptosis,anti-angiogenesis, anti-metastatic activity, anti-adhesion activity,inhibition of heterotypic or homotypic adhesion, and interference insignaling pathways, may also provide a therapeutic effect. Nakedantibodies include polyclonal and monoclonal antibodies, naturallyoccurring or recombinant antibodies, such as chimeric, humanized orhuman antibodies and fragments thereof. In some cases a “naked antibody”may also refer to a “naked” antibody fragment. As defined herein,“naked” is synonymous with “unconjugated,” and means not linked orconjugated to a therapeutic agent.

A chimeric antibody is a recombinant protein that contains the variabledomains of both the heavy and light antibody chains, including thecomplementarity determining regions (CDRs) of an antibody derived fromone species, preferably a rodent antibody, more preferably a murineantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a primate, cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a murine antibody, are transferred fromthe heavy and light variable chains of the murine antibody into humanheavy and light variable domains (framework regions). The constantdomains of the antibody molecule are derived from those of a humanantibody. In some cases, specific residues of the framework region ofthe humanized antibody, particularly those that are touching or close tothe CDR sequences, may be modified, for example replaced with thecorresponding residues from the original murine, rodent, subhumanprimate, or other antibody.

A human antibody is an antibody obtained, for example, from transgenicmice that have been “engineered” to produce human antibodies in responseto antigenic challenge. In this technique, elements of the human heavyand light chain loci are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for various antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, human antibody variable domain genes arecloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, fortheir review, see e.g. Johnson and Chiswell, Current Opinion inStructural Biology 3:5564-571 (1993). Human antibodies may also begenerated by in vitro activated B cells. See U.S. Pat. Nos. 5,567,610and 5,229,275, the Examples section of each of which is incorporatedherein by reference.

A therapeutic agent is an atom, molecule, or compound that is useful inthe treatment of a disease. Examples of therapeutic agents include, butare not limited to, antibodies, antibody fragments, ADCs, drugs,cytotoxic agents, pro-apopoptotic agents, toxins, nucleases (includingDNAses and RNAses), hormones, immunomodulators, chelators, boroncompounds, photoactive agents or dyes, radionuclides, oligonucleotides,interference RNA, siRNA, RNAi, anti-angiogenic agents, chemotherapeuticagents, cyokines, chemokines, prodrugs, enzymes, binding proteins orpeptides or combinations thereof.

An ADC is an antibody, antigen-binding antibody fragment, antibodycomplex or antibody fusion protein that is conjugated to a therapeuticagent. Conjugation may be covalent or non-covalent. Preferably,conjugation is covalent.

As used herein, the term antibody fusion protein is arecombinantly-produced antigen-binding molecule in which one or morenatural antibodies, single-chain antibodies or antibody fragments arelinked to another moiety, such as a protein or peptide, a toxin, acytokine, a hormone, etc. In certain preferred embodiments, the fusionprotein may comprise two or more of the same or different antibodies,antibody fragments or single-chain antibodies fused together, which maybind to the same epitope, different epitopes on the same antigen, ordifferent antigens.

An immunomodulator is a therapeutic agent that when present, alters,suppresses or stimulates the body's immune system. Typically, animmunomodulator of use stimulates immune cells to proliferate or becomeactivated in an immune response cascade, such as macrophages, dendriticcells, B-cells, and/or T-cells. However, in some cases animmunomodulator may suppress proliferation or activation of immunecells. An example of an immunomodulator as described herein is acytokine, which is a soluble small protein of approximately 5-20 kDathat is released by one cell population (e.g., primed T-lymphocytes) oncontact with specific antigens, and which acts as an intercellularmediator between cells. As the skilled artisan will understand, examplesof cytokines include lymphokines, monokines, interleukins, and severalrelated signaling molecules, such as tumor necrosis factor (TNF) andinterferons. Chemokines are a subset of cytokines. Certain interleukinsand interferons are examples of cytokines that stimulate T cell or otherimmune cell proliferation. Exemplary interferons include interferon-α,interferon-β, interferon-γ and interferon-λ.

Anti-Trop-2 Antibodies

Preferably, the subject ADCs include at least one antibody or fragmentthereof that binds to Trop-2. In a specific preferred embodiment, theanti-Trop-2 antibody may be a humanized RS7 antibody (see, e.g., U.S.Pat. No. 7,238,785, incorporated herein by reference in its entirety),comprising the light chain CDR sequences CDR1 (KASQDVSIAVA, SEQ IDNO:14); CDR2 (SASYRYT, SEQ ID NO:15); and CDR3 (QQHYITPLT, SEQ ID NO:16)and the heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:17); CDR2(WINTYTGEPTYTDDFKG, SEQ ID NO:18) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:19).

The RS7 antibody was a murine IgG₁ raised against a crude membranepreparation of a human primary squamous cell lung carcinoma. (Stein etal., Cancer Res. 50:1330, 1990) The RS7 antibody recognizes a 46-48 kDaglycoprotein, characterized as cluster 13. (Stein et al., Int. J. CancerSupp. 8:98-102, 1994) The antigen was designated as EGP-1 (epithelialglycoprotein-1), but is also referred to as Trop-2.

Trop-2 is a type-I transmembrane protein and has been cloned from bothhuman (Fornaro et al., Int J Cancer 1995; 62:610-8) and mouse cells(Sewedy et al., Int J Cancer 1998; 75:324-30). In addition to its roleas a tumor-associated calcium signal transducer (Ripani et al., Int JCancer 1998; 76:671-6), the expression of human Trop-2 was shown to benecessary for tumorigenesis and invasiveness of colon cancer cells,which could be effectively reduced with a polyclonal antibody againstthe extracellular domain of Trop-2 (Wang et al., Mol Cancer Ther 2008;7:280-5).

The growing interest in Trop-2 as a therapeutic target for solid cancers(Cubas et al., Biochim Biophys Acta 2009; 1796:309-14) is attested byfurther reports that documented the clinical significance ofoverexpressed Trop-2 in breast (Huang et al., Clin Cancer Res 2005;11:4357-64), colorectal (Ohmachi et al., Clin Cancer Res 2006;12:3057-63; Fang et al., Int J Colorectal Dis 2009; 24:875-84), and oralsquamous cell (Fong et al., Modern Pathol 2008; 21:186-91) carcinomas.The latest evidence that prostate basal cells expressing high levels ofTrop-2 are enriched for in vitro and in vivo stem-like activity isparticularly noteworthy (Goldstein et al., Proc Natl Acad Sci USA 2008;105:20882-7).

Flow cytometry and immunohistochemical staining studies have shown thatthe RS7 MAb detects antigen on a variety of tumor types, with limitedbinding to normal human tissue (Stein et al., 1990). Trop-2 is expressedprimarily by carcinomas such as carcinomas of the lung, stomach, urinarybladder, breast, ovary, uterus, and prostate. Localization and therapystudies using radiolabeled murine RS7 MAb in animal models havedemonstrated tumor targeting and therapeutic efficacy (Stein et al.,1990; Stein et al., 1991).

Strong RS7 staining has been demonstrated in tumors from the lung,breast, bladder, ovary, uterus, stomach, and prostate. (Stein et al.,Int. J. Cancer 55:938, 1993) The lung cancer cases comprised bothsquamous cell carcinomas and adenocarcinomas. (Stein et al., Int. J.Cancer 55:938, 1993) Both cell types stained strongly, indicating thatthe RS7 antibody does not distinguish between histologic classes ofnon-small-cell carcinoma of the lung.

The RS7 MAb is rapidly internalized into target cells (Stein et al.,1993). The internalization rate constant for RS7 MAb is intermediatebetween the internalization rate constants of two other rapidlyinternalizing MAbs, which have been demonstrated to be useful for ADCproduction. (Id.) It is well documented that internalization of ADCs isa requirement for anti-tumor activity. (Pastan et al., Cell 47:641,1986) Internalization of ADCs has been described as a major factor inanti-tumor efficacy. (Yang et al., Proc. Nat'l Acad. Sci. USA 85:1189,1988) Thus, the RS7 antibody exhibits several important properties fortherapeutic applications.

While the hRS7 antibody is preferred, other anti-Trop-2 antibodies areknown and/or publicly available and in alternative embodiments may beutilized in the subject ADCs. While humanized or human antibodies arepreferred for reduced immunogenicity, in alternative embodiments achimeric antibody may be of use. As discussed below, methods of antibodyhumanization are well known in the art and may be utilized to convert anavailable murine or chimeric antibody into a humanized form.

Anti-Trop-2 antibodies are commercially available from a number ofsources and include LS-C126418, LS-C178765, LS-C126416, LS-C126417(LifeSpan BioSciences, Inc., Seattle, Wash.); 10428-MM01, 10428-MM02,10428-R001, 10428-R030 (Sino Biological Inc., Beijing, China); MR54(eBioscience, San Diego, Calif.); sc-376181, sc-376746, Santa CruzBiotechnology (Santa Cruz, Calif.); MM0588-49D6, (Novus Biologicals,Littleton, Colo.); ab79976, and ab89928 (ABCAM®, Cambridge, Mass.).

Other anti-Trop-2 antibodies have been disclosed in the patentliterature. For example, U.S. Publ. No. 2013/0089872 disclosesanti-Trop-2 antibodies K5-70 (Accession No. FERM BP-11251), K5-107(Accession No. FERM BP-11252), K5-116-2-1 (Accession No. FERM BP-11253),T6-16 (Accession No. FERM BP-11346), and T5-86 (Accession No. FERMBP-11254), deposited with the International Patent Organism Depositary,Tsukuba, Japan. U.S. Pat. No. 5,840,854 disclosed the anti-Trop-2monoclonal antibody BR110 (ATCC No. HB11698). U.S. Pat. No. 7,420,040disclosed an anti-Trop-2 antibody produced by hybridoma cell lineAR47A6.4.2, deposited with the IDAC (International Depository Authorityof Canada, Winnipeg, Canada) as accession number 141205-05. U.S. Pat.No. 7,420,041 disclosed an anti-Trop-2 antibody produced by hybridomacell line AR52A301.5, deposited with the IDAC as accession number141205-03. U.S. Publ. No. 2013/0122020 disclosed anti-Trop-2 antibodies3E9, 6G11, 7E6, 15E2, 18B1. Hybridomas encoding a representativeantibody were deposited with the American Type Culture Collection(ATCC), Accession Nos. PTA-12871 and PTA-12872. Immunoconjugate PF06263507, comprising an anti-5T4 (anti-Trop-2) antibody attached to thetubulin inhibitor monomethylauristatin F (MMAF) is available fromPfizer, Inc. (Groton, Conn.) (see, e.g., Sapra et al., 2013, Mol CancerTher 12:38-47). U.S. Pat. No. 8,715,662 discloses anti-Trop-2 antibodiesproduced by hybridomas deposited at the AID-ICLC (Genoa, Italy) withdeposit numbers PD 08019, PD 08020 and PD 08021. U.S. Patent ApplicationPubl. No. 20120237518 discloses anti-Trop-2 antibodies 77220, KM4097 andKM4590. U.S. Pat. No. 8,309,094 (Wyeth) discloses antibodies A1 and A3,identified by sequence listing. The Examples section of each patent orpatent application cited above in this paragraph is incorporated hereinby reference. Non-patent publication Lipinski et al. (1981, Proc Natl.Acad Sci USA, 78:5147-50) disclosed anti-Trop-2 antibodies 162-25.3 and162-46.2.

Numerous anti-Trop-2 antibodies are known in the art and/or publiclyavailable. As discussed below, methods for preparing antibodies againstknown antigens were routine in the art. The sequence of the human Trop-2protein was also known in the art (see, e.g., GenBank Accession No.CAA54801.1). Methods for producing humanized, human or chimericantibodies were also known. The person of ordinary skill, reading theinstant disclosure in light of general knowledge in the art, would havebeen able to make and use the genus of anti-Trop-2 antibodies in thesubject ADCs.

Use of antibodies against targets related to Trop-2 has been disclosedfor immunotherapeutics other than ADCs. The murine anti-Trop-1 IgG2aantibody edrecolomab (PANOREX®) has been used for treatment ofcolorectal cancer, although the murine antibody is not well suited forhuman clinical use (Baeuerle & Gires, 2007, Br. J Cancer 96:417-423).Low-dose subcutaneous administration of ecrecolomab was reported toinduce humoral immune responses against the vaccine antigen (Baeuerle &Gires, 2007). Adecatumumab (MT201), a fully human anti-Trop-1 antibody,has been used in metastatic breast cancer and early-stage prostatecancer and is reported to act through ADCC and CDC activity (Baeuerle &Gires, 2007). MT110, a single-chain anti-Trop-1/anti-CD3 bispecificantibody construct has reported efficacy against ovarian cancer(Baeuerle & Gires, 2007). Proxinium, an immunotoxin comprisinganti-Trop-1 single-chain antibody fused to Pseudomonas exotoxin, hasbeen tested in head-and-neck and bladder cancer (Baeuerle & Gires,2007). None of these studies contained any disclosure of the use ofanti-Trop-2 ADCs.

ABCG2 Inhibitors

In preferred embodiments, combination therapy is performed with ananti-Trop-2 ADC (e.g., hRS7-CL2A-SN-38) in combination with one or moreinhibitors of an ABC transporter, preferably an inhibitor of ABCB1,ABCC1 or ABCG2, more preferably an inhibitor of ABCG2. The role of ABCG2inhibitors in combination cancer therapy has been recently reviewed byRicci et al. (2015, J Develop Drugs 4:138). ABCG2 is unique among theABC transporters in that it is mainly overexpressed in drug-resistantsolid tumors, although it has also been found to be overexpressed in anumber of hematopoietic tumors along with ABCB1 and ABCC1 (Ricci et al.,2015, J Develop Drugs 4:138). Although ABCG2 can transport a number ofchemotherapeutic agents, the most well known include topotecan,mitoxantrone, SN-38, doxorubicin and daunorubicin (Ricci et al., 2015, JDevelop Drugs 4:138). Elevated expression of ABCG2 has been reported tobe associated with decreased survival rates in small cell lung cancer,non-small cell lung cancer, pancreatic cancer, mantle cell lymphoma,acute myeloid leukemia, ovarian cancer, colorectal cancer and breastcancer (Ricci et al., 2015, J Develop Drugs 4:138).

Many drugs have been found to be inhibitors of ABCG2 activity (see Table1). However, of these, only a handful have been tested in vivo and/or inhumans, with relatively limited success to date in improvingchemotherapeutic efficacy (Ricci et al., 2015, J Develop Drugs 4:138).

TABLE 1 ABCG2 Inhibitors With In vitro Efficacy* in Clinical Drug vivotrials 1,4-dihydropyridines[48] Artesunate[39] X AST1306[51]Bifendate-chalcone hybrids[53] Botryllamides[55] Cadmium[57] CalciumChannel Blockers (nicardipine, nitrendipine, nimodipine,dipyridamole)[59] Camptothecin analog (ST1481)[40] X Camptothecin analog(CHO793076)[41] X Cannabinoids[63] CCT129202[42] X Chalcone[66]Curcumin[30] X Cyclosporin A[69] Dihydropyridines and Pyridines[71] XDimethoxyaurones[73] Dofequidar fumarate[38] X Repurposed Drugs[76] EGFRInhibitors[78] Flavones & Benzoflavones[80] Tropical PlantFlavonoids[82] Fruit Juices (quercetin, kaempferol, bergamotin,6′,7′-dihydroxybergamottin, tangeretin, nobiletin, hesperidin,hesperetin)[84] Fumitremorgin C[29, 31] X Fumitremorgin C analogue(ko143)[32] X Gefitinib[44, 88] X GF120918, BNP1350[32, 33] X X GW583340and GW2974[91] HM30181 Derivatives[93] Human cathelicidin[95] Imatinibmesylate[43, 98] X X Lapatinib[45-47, 49] X X LY294002[50] MBLI-87[52] XMethoxy Stilbenes[54] Mithramycin A[56] Quercetin derivatives[58]Naphthopyrones[60] Nilotinib[61] Novobiocin[62] X NP-1250[64] OlomoucineII and purvalanol A[65] Organ chlorine and Pyrethroid[67] OSI-930[68]Phytoestrogens/Flavonoids[70] Piperazinobenzopyranones andPhenalkylaminobenzopyranones[72] Ponatinib[74] PZ-39[75]Quinazolines[77] Quizartinib[79] Sildenafil[81] Sorafenib[83]Substituted Chromones[85] Sunitinib[86] Tandutinib[87] Tariquidar[89]Terpenoids[90] CI1033[92] Toremifene[94] XR9577[96], WK-X-34[96, 97], XWK-X-50[96], and WK-X-84[96] YHO-13177[34]and YHO-13351[34] X *FromRicci et al., 2015, J Develop Drugs 4:138

Fumitremorgin C was the first ABCG2 inhibitor to be described whichreversed chemoresistance of colon carcinoma to MTX (Rabindran et al.,1998, Cancer Res 58:5850-58). Since that time, over 60 agents have beendescribed that inhibit the action of ABCG2 in vitro (Table 1). Of those,only 15 compounds inhibiting ABCG2 activity have exhibited anti-canceractivity in vivo in animal models of human cancer xenografts (Table 1and 2). Only 6 of those compounds are direct antagonists specific forABCG2: curcumin, FTC, Ko143, GF120918 (Elacridar), YHO-13177, YHO-13351,and the recently reported compounds 177, 724, and 505 (Shukla et al.,2009, Pharm Res 26:480-87; Garimella et al., 2005, Cancer ChemotherPharmacol 55:101-9; Allen et al., 2002, Mol Cancer Ther 1:417-25; Hyafilet al., Cancer Res 53:4595-602; Yamazaki et al., 2011, Mol Cancer Ther10:1252-63; Strouse et al., 2013, J Biomol Screen 18:26-38; Strouse etal., 2013, Anal Biochem 437:77-87). Of the specific ABCG2 antagonists,only YHO-13177 and the recently reported compounds CID44640177,CID1434724, and CID46245505 (Ricci et al., 2016, Mol Cancer Ther15:2853-62) were reported to have an antitumor effect when combined withTPT (Ricci et al., 2015, J Develop Drugs 4:138).

A number of compounds have shown efficacy in animal models of cancer(Table 2) (Ricci et al., 2015, J Develop Drugs 4:138). Some of thesecompounds have an indirect inhibitory effect on ABCG2, and includeantimalarial agents, chemotherapeutic analogs of camptothecin, and anaurora kinase inhibitor (Table 2) (Ricci et al., 2015, J Develop Drugs4:138). The aurora kinase inhibitor, CCT129202, was able to increase theaccumulation of doxorubicin and rhodamine 123 in ABCB1 and ABCG2overexpressing human colon cancer cells.

As discussed in the Examples below, the present studies demonstrate thatat least the ABCG2 inhibitors fumitremorgin C, Ko143 and YHO-13351restored toxicity of SN-38 in MDA-MB-231 human breast cancer cells andNCI-N87-S120 human gastric cancer cells with induced resistance toSN-38, and the combination of YHO-13351 with IMMU-132 (anti-Trop-2 ADC)increased median survival of mice bearing NCI-N87-S120 xenografts. Theseresults support the use of ABCG2 inhibitors with anti-cancer ADCs,preferably anti-Trop-2 ADCs, more preferably IMMU-132, for combinationtherapy in drug resistant cancers. Such combination therapy may beeffective in either cancers with innate resistance to chemotherapeuticagents or in cancers that develop resistance during the course oftherapy. The instant invention demonstrates unexpected efficacy ofcombination therapy with ABCG2 inhibitors and anti-cancer ADCs.

TABLE 2 Compounds Antagonizing ABCG2 In vivo In Animal Cancer Models*Drug Mechanism of Action Cancer Artesunate[39] Down-regulates expressionof NSCLC ABCG2 Camptothecin analog Chemotherapy Colon Carcinoma(ST1481)[40] Camptothecin analog Chemotherapy Ovarian and Breast Cancer(CHO793076)[41] CCT129202[42] Aurora Kinase inhibitor Colon CarcinomaCurcumin[30] ABCG2 inhibitor Breast Cancer Dihydropyridines and ABCB1 +ABCG2 inhibitor Breast Cancer and NSCLC Pyridines[71] Dofequidarfumarate[38] ABCB1, ABCC1, ABCG2 HeLa side population cancer stem-likeinhibitor cells Fumitremorgin C[31] ABCG2 inhibitor Ovarian CancerFumitremorginC ABCG2 inhibitor Ovarian Cancer analogue(ko143)[32]Gefitinib[44] ERBB1 inhibitor, ABCG2 Neuroblastoma, Osteosarcoma,inhibitor Rhabdomyosarcoma, Glioblastoma GF120918[33], BNP1350[33] ABCG2inhibitor Mouse Leukemia and Ovarian Cancer Imatinib mesylate[43] PDGFRinhibitor, ABCG2 Rhabdomyosarcoma inhibitor Lapatinib[45] Tyrosinekinase inhibitor, Vincristine resistant epidermoid ABCB1, ABCG2inhibitor carcinoma WK-X-34[97] ABCB1 and ABCG2 inhibitor Ovarian CancerYHO-13177[34]and ABCG2 inhibitor Human colon cancer, mouse leukemiaYHO-13351[34] *From Ricci et al., 2015, J Develop Drugs 4:138General Antibody Techniques

Techniques for preparing monoclonal antibodies against virtually anytarget antigen are well known in the art. See, for example, Köhler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991). Briefly, monoclonal antibodies can be obtained by injecting micewith a composition comprising an antigen, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures. The person of ordinary skill will realize that whereantibodies are to be administered to human subjects, the antibodies willbind to human antigens.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A or Protein-G Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art, as discussedbelow.

The skilled artisan will realize that the claimed methods andcompositions may utilize any of a wide variety of antibodies known inthe art. Antibodies of use may be commercially obtained from a widevariety of known sources. For example, a variety of antibody secretinghybridoma lines are available from the American Type Culture Collection(ATCC, Manassas, Va.). A large number of antibodies against variousdisease targets, including but not limited to tumor-associated antigens,have been deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206; 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953;5,525,338, the Examples section of each of which is incorporated hereinby reference. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art. Isolated antibodies may be conjugated totherapeutic agents, such as camptothecins, using the techniquesdisclosed herein.

Chimeric and Humanized Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. Methods for constructingchimeric antibodies are well known in the art (e.g., Leung et al., 1994,Hybridoma 13:469).

A chimeric monoclonal antibody may be humanized by transferring themouse CDRs from the heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. The mouse framework regions (FR) in the chimeric monoclonalantibody are also replaced with human FR sequences. To preserve thestability and antigen specificity of the humanized monoclonal, one ormore human FR residues may be replaced by the mouse counterpartresidues. Humanized monoclonal antibodies may be used for therapeutictreatment of subjects. Techniques for production of humanized monoclonalantibodies are well known in the art. (See, e.g., Jones et al., 1986,Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen etal., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad.Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest etal., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993,150:2844.)

Other embodiments may concern non-human primate antibodies. Generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., WO 91/11465 (1991), and inLosman et al., Int. J. Cancer 46: 310 (1990). In another embodiment, anantibody may be a human monoclonal antibody. Such antibodies may beobtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge, asdiscussed below.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50; each incorporated herein by reference). Such fully humanantibodies are expected to exhibit even fewer side effects than chimericor humanized antibodies and to function in vivo as essentiallyendogenous human antibodies. In certain embodiments, the claimed methodsand procedures may utilize human antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as cancer (Dantas-Barbosa et al., 2005). Theadvantage to constructing human antibodies from a diseased individual isthat the circulating antibody repertoire may be biased towardsantibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.) Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.) RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J Mol. Biol. 222:581-97,incorporated herein by reference). Library construction was performedaccording to Andris-Widhopf et al. (2000, In: Phage Display LaboratoryManual, Barbas et al. (eds), 1s^(t) edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22, incorporatedherein by reference). The final Fab fragments were digested withrestriction endonucleases and inserted into the bacteriophage genome tomake the phage display library. Such libraries may be screened bystandard phage display methods. The skilled artisan will realize thatthis technique is exemplary only and any known method for making andscreening human antibodies or antibody fragments by phage display may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. Methods for obtaining humanantibodies from transgenic mice are described by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994). A non-limiting example of such a systemis the XENOMOUSE® (e.g., Green et al., 1999, J. Immunol. Methods231:11-23, incorporated herein by reference) from Abgenix (Fremont,Calif.), in which the mouse antibody genes have been inactivated andreplaced by functional human antibody genes, while the remainder of themouse immune system remains intact.

The transgenic mice were transformed with germline-configured YACs(yeast artificial chromosomes) that contained portions of the human IgHand Ig kappa loci, including the majority of the variable regionsequences, along accessory genes and regulatory sequences. The humanvariable region repertoire may be used to generate antibody producing Bcells, which may be processed into hybridomas by known techniques. AXENOMOUSE® immunized with a target antigen will produce human antibodiesby the normal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of geneticallyengineered mice are available, each of which is capable of producing adifferent class of antibody. Transgenically produced human antibodieshave been shown to have therapeutic potential, while retaining thepharmacokinetic properties of normal human antibodies (Green et al.,1999). The skilled artisan will realize that the claimed compositionsand methods are not limited to use of the XENOMOUSE® system but mayutilize any transgenic animal that has been genetically engineered toproduce human antibodies.

Production of Antibody Fragments

Some embodiments of the claimed methods and/or compositions may concernantibody fragments. Such antibody fragments may be obtained, forexample, by pepsin or papain digestion of whole antibodies byconventional methods. For example, antibody fragments may be produced byenzymatic cleavage of antibodies with pepsin to provide a 5S fragmentdenoted F(ab′)₂. This fragment may be further cleaved using a thiolreducing agent and, optionally, a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce 3.5SFab′ monovalent fragments. Alternatively, an enzymatic cleavage usingpepsin produces two monovalent Fab fragments and an Fc fragment.Exemplary methods for producing antibody fragments are disclosed in U.S.Pat. Nos. 4,036,945; 4,331,647; Nisonoff et al., 1960, Arch. Biochem.Biophys., 89:230; Porter, 1959, Biochem. J., 73:119; Edelman et al.,1967, METHODS IN ENZYMOLOGY, page 422 (Academic Press), and Coligan etal. (eds.), 1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments or other enzymatic, chemical or genetic techniques also may beused, so long as the fragments bind to the antigen that is recognized bythe intact antibody. For example, Fv fragments comprise an associationof V_(H) and V_(L) chains. This association can be noncovalent, asdescribed in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69:2659.Alternatively, the variable chains may be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde. SeeSandhu, 1992, Crit. Rev. Biotech., 12:437.

Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (scFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains, connected by an oligonucleotideslinker sequence. The structural gene is inserted into an expressionvector that is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are well-known in the art. See Whitlow et al., 1991, Methods: ACompanion to Methods in Enzymology 2:97; Bird et al., 1988, Science,242:423; U.S. Pat. No. 4,946,778; Pack et al., 1993, Bio/Technology,11:1271, and Sandhu, 1992, Crit. Rev. Biotech., 12:437.

Another form of an antibody fragment is a single-domain antibody (dAb),sometimes referred to as a single chain antibody. Techniques forproducing single-domain antibodies are well known in the art (see, e.g.,Cossins et al., Protein Expression and Purification, 2007, 51:253-59;Shuntao et al., Molec Immunol 2006, 43:1912-19; Tanha et al., J. Biol.Chem. 2001, 276:24774-780). Other types of antibody fragments maycomprise one or more complementarity-determining regions (CDRs). CDRpeptides (“minimal recognition units”) can be obtained by constructinggenes encoding the CDR of an antibody of interest. Such genes areprepared, for example, by using the polymerase chain reaction tosynthesize the variable region from RNA of antibody-producing cells. SeeLarrick et al., 1991, Methods: A Companion to Methods in Enzymology2:106; Ritter et al. (eds.), 1995, MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, pages 166-179 (CambridgeUniversity Press); Birch et al., (eds.), 1995, MONOCLONAL ANTIBODIES:PRINCIPLES AND APPLICATIONS, pages 137-185 (Wiley-Liss, Inc.)

Antibody Variations

In certain embodiments, the sequences of antibodies, such as the Fcportions of antibodies, may be varied to optimize the physiologicalcharacteristics of the conjugates, such as the half-life in serum.Methods of substituting amino acid sequences in proteins are widelyknown in the art, such as by site-directed mutagenesis (e.g. Sambrook etal., Molecular Cloning, A laboratory manual, 2^(nd) Ed, 1989). Inpreferred embodiments, the variation may involve the addition or removalof one or more glycosylation sites in the Fc sequence (e.g., U.S. Pat.No. 6,254,868, the Examples section of which is incorporated herein byreference). In other preferred embodiments, specific amino acidsubstitutions in the Fc sequence may be made (e.g., Hornick et al.,2000, J Nucl Med 41:355-62; Hinton et al., 2006, J Immunol 176:346-56;Petkova et al. 2006, Int Immunol 18:1759-69; U.S. Pat. No. 7,217,797;each incorporated herein by reference).

Target Antigens and Exemplary Antibodies

In a preferred embodiment, antibodies are used that recognize and/orbind to antigens that are expressed at high levels on target cells andthat are expressed predominantly or exclusively on diseased cells versusnormal tissues. More preferably, the antibodies internalize rapidlyfollowing binding. An exemplary rapidly internalizing antibody is theLL1 (anti-CD74) antibody, with a rate of internalization ofapproximately 8×10⁶ antibody molecules per cell per day (e.g., Hansen etal., 1996, Biochem J. 320:293-300). Thus, a “rapidly internalizing”antibody may be one with an internalization rate of about 1×10⁶ to about1×10⁷ antibody molecules per cell per day. Antibodies of use in theclaimed compositions and methods may include MAbs with properties asrecited above. Exemplary antibodies of use for therapy of, for example,cancer include but are not limited to LL1 (anti-CD74), LL2 or RFB4(anti-CD22), veltuzumab (hA20, anti-CD20), rituxumab (anti-CD20),obinutuzumab (GA101, anti-CD20), lambrolizumab (anti-PD-1 receptor),nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4), RS7(anti-Trop-2), PAM4 (anti-MUC-5ac), MN-14 (anti-CEACAM-5), MN-15 or MN-3(anti-CEACAM-6), Mu-9 (anti-colon-specific antigen-p), Immu 31 (ananti-alpha-fetoprotein), hR1 (anti-IGF-1R), A19 (anti-CD19), TAG-72(e.g., CC49), Tn, J591 or HuJ591 (anti-PSMA (prostate-specific membraneantigen)), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA), G250 (ananti-carbonic anhydrase IX MAb), L243 (anti-HLA-DR) alemtuzumab(anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab(anti-CD33), ibritumomab tiuxetan (anti-CD20); panitumumab (anti-EGFR);tositumomab (anti-CD20); and trastuzumab (anti-ErbB2). Such antibodiesare known in the art (e.g., U.S. Pat. Nos. 5,686,072; 5,874,540;6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300; 6,899,864;6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785; 7,238,786;7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491; 7,612,180;7,642,239; and U.S. Patent Application Publ. No. 20050271671;20060193865; 20060210475; 20070087001; the Examples section of eachincorporated herein by reference.) Specific known antibodies of useinclude hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,251,164),hA19 (U.S. Pat. No. 7,109,304), hIMMU-31 (U.S. Pat. No. 7,300,655), hLL1(U.S. Pat. No. 7,312,318), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S.Pat. No. 7,387,773), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat.No. 6,676,924), hMN-15 (U.S. Pat. No. 7,541,440), hR1 (U.S. patentapplication Ser. No. 12/772,645), hRS7 (U.S. Pat. No. 7,238,785), hMN-3(U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S. patent application Ser.No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO2009/130575) the text of each recited patent or application isincorporated herein by reference with respect to the Figures andExamples sections. In a particularly preferred embodiment, the antibodyis hRS7.

Other useful antigens that may be targeted using the describedconjugates include carbonic anhydrase IX, B7, CCL19, CCL21, CSAp,HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5 MAbs), CD21, CD22, CD23,CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45,CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83,CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM6, CTLA-4,alpha-fetoprotein (AFP), VEGF (e.g., bevacizumab, fibronectin splicevariant), ED-B fibronectin (e.g., L19), EGP-1 (Trop-2), EGP-2 (e.g.,17-1A), EGF receptor (ErbB1) (e.g., cetuximab), ErbB2, ErbB3, Factor H,FHL-1, Flt-3, folate receptor, Ga 733, GRO-β, HMGB-1, hypoxia induciblefactor (HIF), HM1.24, HER-2/neu, insulin-like growth factor (ILGF),IFN-γ, IFN-α, IFN-β, IFN-λ, IL-2R, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R,IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10,IGF-1R, Ia, HM1.24, gangliosides, HCG, the HLA-DR antigen to which L243binds, CD66 antigens, i.e., CD66a-d or a combination thereof, MAGE,mCRP, MCP-1, MIP-1A, MIP-1B, macrophage migration-inhibitory factor(MIF), MUC1, MUC2, MUC3, MUC4, MUC5ac, placental growth factor (P1GF),PSA (prostate-specific antigen), PSMA, PD-1 receptor, NCA-95, NCA-90,A3, A33, Ep-CAM, KS-1, Le(y), mesothelin, 5100, tenascin, TAC, Tnantigen, Thomas-Friedenreich antigens, tumor necrosis antigens, tumorangiogenesis antigens, TNF-α, TRAIL receptor (R1 and R2), Trop-2, VEGFR,RANTES, T101, as well as cancer stem cell antigens, complement factorsC3, C3a, C3b, C5a, C5, and an oncogene product.

A comprehensive analysis of suitable antigen (Cluster Designation, orCD) targets on hematopoietic malignant cells, as shown by flow cytometryand which can be a guide to selecting suitable antibodies fordrug-conjugated immunotherapy, is Craig and Foon, Blood prepublishedonline Jan. 15, 2008; DOL 10.1182/blood-2007-11-120535.

The CD66 antigens consist of five different glycoproteins with similarstructures, CD66a-e, encoded by the carcinoembryonic antigen (CEA) genefamily members, BCG, CGM6, NCA, CGMI and CEA, respectively. These CD66antigens (e.g., CEACAM6) are expressed mainly in granulocytes, normalepithelial cells of the digestive tract and tumor cells of varioustissues. Also included as suitable targets for cancers are cancer testisantigens, such as NY-ESO-1 (Theurillat et al., Int. J. Cancer 2007;120(11):2411-7), as well as CD79a in myeloid leukemia (Kozlov et al.,Cancer Genet. Cytogenet. 2005; 163(1):62-7) and also B-cell diseases,and CD79b for non-Hodgkin's lymphoma (Poison et al., Blood110(2):616-623). A number of the aforementioned antigens are disclosedin U.S. Provisional Application Ser. No. 60/426,379, entitled “Use ofMulti-specific, Non-covalent Complexes for Targeted Delivery ofTherapeutics,” filed Nov. 15, 2002. Cancer stem cells, which areascribed to be more therapy-resistant precursor malignant cellpopulations (Hill and Penis, J. Natl. Cancer Inst. 2007; 99:1435-40),have antigens that can be targeted in certain cancer types, such asCD133 in prostate cancer (Maitland et al., Ernst Schering Found. Sympos.Proc. 2006; 5:155-79), non-small-cell lung cancer (Donnenberg et al., J.Control Release 2007; 122(3):385-91), and glioblastoma (Beier et al.,Cancer Res. 2007; 67(9):4010-5), and CD44 in colorectal cancer (Dalerbaer al., Proc. Natl. Acad. Sci. USA 2007; 104(24)10158-63), pancreaticcancer (Li et al., Cancer Res. 2007; 67(3):1030-7), and in head and necksquamous cell carcinoma (Prince et al., Proc. Natl. Acad. Sci. USA 2007;104(3)973-8). Another useful target for breast cancer therapy is theLIV-1 antigen described by Taylor et al. (Biochem. J. 2003; 375:51-9).The CD47 antigen is a further useful target for cancer stem cells (see,e.g., Naujokat et al., 2014, Immunotherapy 6:290-308; Goto et al., 2014,Eur J Cancer 50:1836-46; Unanue, 2013, Proc Natl Acad Sci USA110:10886-7).

For multiple myeloma therapy, suitable targeting antibodies have beendescribed against, for example, CD38 and CD138 (Stevenson, Mol Med 2006;12(11-12):345-346; Tassone et al., Blood 2004; 104(12):3688-96), CD74(Stein et al., ibid.), CS1 (Tai et al., Blood 2008; 112(4):1329-37, andCD40 (Tai et al., 2005; Cancer Res. 65(13):5898-5906).

Checkpoint inhibitor antibodies have been used in cancer therapy. Immunecheckpoints refer to inhibitory pathways in the immune system that areresponsible for maintaining self-tolerance and modulating the degree ofimmune system response to minimize peripheral tissue damage. However,tumor cells can also activate immune system checkpoints to decrease theeffectiveness of immune response against tumor tissues. Exemplarycheckpoint inhibitor antibodies against cytotoxic T-lymphocyte antigen 4(CTLA4, also known as CD152), programmed cell death protein 1 (PD1, alsoknown as CD279) and programmed cell death 1 ligand 1 (PD-L1, also knownas CD274), may be used in combination with one or more other agents toenhance the effectiveness of immune response against disease cells.Exemplary anti-PD1 antibodies include lambrolizumab (MK-3475, MERCK),nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), AMP-224 (MERCK), andpidilizumab (CT-011, CURETECH LTD.). Anti-PD1 antibodies arecommercially available, for example from ABCAM® (AB137132), BIOLEGEND®(EH12.2H7, RMP1-14) and AFFYMETRIX EBIOSCIENCE (J105, J116, MIH4).Exemplary anti-PD-L1 antibodies include MDX-1105 (MEDAREX), MEDI4736(MEDIMMUNE) MPDL3280A (GENENTECH) and BMS-936559 (BRISTOL-MYERS SQUIBB).Anti-PD-L1 antibodies are also commercially available, for example fromAFFYMETRIX EBIOSCIENCE (MIH1). Exemplary anti-CTLA4 antibodies includeipilimumab (Bristol-Myers Squibb) and tremelimumab (PFIZER). Anti-PD1antibodies are commercially available, for example from ABCAM®(AB134090), SINO BIOLOGICAL INC. (11159-H03H, 11159-H08H), and THERMOSCIENTIFIC PIERCE (PAS-29572, PAS-23967, PAS-26465, MA1-12205,MA1-35914). Ipilimumab has recently received FDA approval for treatmentof metastatic melanoma (Wada et al., 2013, J Transl Med 11:89).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, colon and chronic lymphocytic leukemia(e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran,2011, LeukLymphoma 52:1446-54); autoimmune diseases such as rheumatoidarthritis and systemic lupus erythematosus (Morand & Leech, 2005, FrontBiosci 10:12-22; Shachar & Haran, 2011, LeukLymphoma 52:1446-54); kidneydiseases such as renal allograft rejection (Lan, 2008, Nephron ExpNephrol. 109:e79-83); and numerous inflammatory diseases (Meyer-Siegleret al., 2009, Mediators Inflamm epub Mar. 22, 2009; Takahashi et al.,2009, Respir Res 10:33; Milatuzumab (hLL1) is an exemplary anti-CD74antibody of therapeutic use for treatment of MIF-mediated diseases.

Anti-TNF-α antibodies are known in the art and may be of use to treatimmune diseases, such as autoimmune disease, immune dysfunction (e.g.,graft-versus-host disease, organ transplant rejection) or diabetes.Known antibodies against TNF-α include the human antibody CDP571 (Ofeiet al., 2011, Diabetes 45:881-85); murine antibodies MTNFAI, M2TNFAI,M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, Ill.);infliximab (Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and manyother known anti-TNF-α antibodies may be used in the claimed methods andcompositions. Other antibodies of use for therapy of immunedysregulatory or autoimmune disease include, but are not limited to,anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab orhL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25);daclizumab (anti-CD25); efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3receptor); anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-a4integrin) and omalizumab (anti-IgE).

In another preferred embodiment, antibodies are used that internalizerapidly and are then re-expressed, processed and presented on cellsurfaces, enabling continual uptake and accretion of circulatingconjugate by the cell. An example of a most-preferred antibody/antigenpair is LL1, an anti-CD74 MAb (invariant chain, class II-specificchaperone, Ii) (see, e.g., U.S. Pat. Nos. 6,653,104; 7,312,318; theExamples section of each incorporated herein by reference). The CD74antigen is highly expressed on B-cell lymphomas (including multiplemyeloma) and leukemias, certain T-cell lymphomas, melanomas, colonic,lung, and renal cancers, glioblastomas, and certain other cancers (Onget al., Immunology 98:296-302 (1999)). A review of the use of CD74antibodies in cancer is contained in Stein et al., Clin Cancer Res. 2007Sep. 15; 13(18 Pt 2):55565-5563s, incorporated herein by reference.

The diseases that are preferably treated with anti-CD74 antibodiesinclude, but are not limited to, non-Hodgkin's lymphoma, Hodgkin'sdisease, melanoma, lung, renal, colonic cancers, glioblastomemultiforme, histiocytomas, myeloid leukemias, and multiple myeloma.Continual expression of the CD74 antigen for short periods of time onthe surface of target cells, followed by internalization of the antigen,and re-expression of the antigen, enables the targeting LL1 antibody tobe internalized along with any chemotherapeutic moiety it carries. Thisallows a high, and therapeutic, concentration of LL1-chemotherapeuticdrug conjugate to be accumulated inside such cells. InternalizedLL1-chemotherapeutic drug conjugates are cycled through lysosomes andendosomes, and the chemotherapeutic moiety is released in an active formwithin the target cells.

Antibodies of use to treat autoimmune disease or immune systemdysfunctions (e.g., graft-versus-host disease, organ transplantrejection) are known in the art and may be conjugated to SN-38 using thedisclosed methods and compositions. Antibodies of use to treatautoimmune/immune dysfunction disease may bind to exemplary antigensincluding, but not limited to, BCL-1, BCL-2, BCL-6, CD1a, CD2, CD3, CD4,CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20,CD21, CD22, CD23, CD25, CD33, CD34, CD38, CD40, CD40L, CD41a, CD43,CD45, CD55, TNF-alpha, interferon and HLA-DR.

Antibodies that bind to these and other target antigens, discussedabove, may be used to treat autoimmune or immune dysfunction diseases.Autoimmune diseases that may be treated with ADCs may include acuteidiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenicpurpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemiclupus erythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, ANCA-associated vasculitides, Addison's disease, rheumatoidarthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

The antibodies discussed above and other known antibodies againstdisease-associated antigens may be used as camptothecin-conjugates, morepreferably SN-38-conjugates, in the practice of the claimed methods andcompositions. In a most preferred embodiment, the drug-conjugatedantibody is an anti-Trop-2-SN-38 (e.g., hRS7-SN-38) conjugate.

Bispecific and Multispecific Antibodies

Bispecific antibodies are useful in a number of biomedical applications.For instance, a bispecific antibody with binding sites for a tumor cellsurface antigen and for a T-cell surface receptor can direct the lysisof specific tumor cells by T cells. Bispecific antibodies recognizinggliomas and the CD3 epitope on T cells have been successfully used intreating brain tumors in human patients (Nitta, et al. Lancet. 1990;355:368-371). A preferred bispecific antibody is an anti-CD3 X anti-CD19antibody. In alternative embodiments, an anti-CD3 antibody or fragmentthereof may be attached to an antibody or fragment against anotherB-cell associated antigen, such as anti-CD3 X anti-Trop-2, anti-CD3 Xanti-CD20, anti-CD3 X anti-CD22, anti-CD3 X anti-HLA-DR or anti-CD3 Xanti-CD74. In certain embodiments, the techniques and compositions fortherapeutic agent conjugation disclosed herein may be used withbispecific or multispecific antibodies as the targeting moieties.

Numerous methods to produce bispecific or multispecific antibodies areknown, as disclosed, for example, in U.S. Pat. No. 7,405,320, theExamples section of which is incorporated herein by reference.Bispecific antibodies can be produced by the quadroma method, whichinvolves the fusion of two different hybridomas, each producing amonoclonal antibody recognizing a different antigenic site (Milstein andCuello, Nature, 1983; 305:537-540).

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies (Staerz, et al. Nature, 1985; 314:628-631; Perez,et al. Nature, 1985; 316:354-356). Bispecific antibodies can also beproduced by reduction of each of two parental monoclonal antibodies tothe respective half molecules, which are then mixed and allowed toreoxidize to obtain the hybrid structure (Staerz and Bevan. Proc NatlAcad Sci USA. 1986; 83:1453-1457). Another alternative involveschemically cross-linking two or three separately purified Fab′ fragmentsusing appropriate linkers. (See, e.g., European Patent Application0453082).

Other methods include improving the efficiency of generating hybridhybridomas by gene transfer of distinct selectable markers viaretrovirus-derived shuttle vectors into respective parental hybridomas,which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA.1990, 87:2941-2945); or transfection of a hybridoma cell line withexpression plasmids containing the heavy and light chain genes of adifferent antibody.

Cognate V_(H) and V_(L) domains can be joined with a peptide linker ofappropriate composition and length (usually consisting of more than 12amino acid residues) to form a single-chain Fv (scFv) with bindingactivity. Methods of manufacturing scFvs are disclosed in U.S. Pat. Nos.4,946,778 and 5,132,405, the Examples section of each of which isincorporated herein by reference. Reduction of the peptide linker lengthto less than 12 amino acid residues prevents pairing of V_(H) and V_(L)domains on the same chain and forces pairing of V_(H) and V_(L) domainswith complementary domains on other chains, resulting in the formationof functional multimers. Polypeptide chains of V_(H) and V_(L) domainsthat are joined with linkers between 3 and 12 amino acid residues formpredominantly dimers (termed diabodies). With linkers between 0 and 2amino acid residues, trimers (termed triabody) and tetramers (termedtetrabody) are favored, but the exact patterns of oligomerization appearto depend on the composition as well as the orientation of V-domains(V_(H)-linker-V_(L) or V_(L)-linker-V_(H)), in addition to the linkerlength.

These techniques for producing multispecific or bispecific antibodiesexhibit various difficulties in terms of low yield, necessity forpurification, low stability or the labor-intensiveness of the technique.More recently, a technique known as “dock and lock” (DNL®) has beenutilized to produce combinations of virtually any desired antibodies,antibody fragments and other effector molecules (see, e.g., U.S. Pat.Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070;7,871,622; 7,906,121; 7,906,118; 8,163,291; 7,901,680; 7,981,398;8,003,111 and 8,034,352, the Examples section of each of whichincorporated herein by reference). The technique utilizes complementaryprotein binding domains, referred to as anchoring domains (AD) anddimerization and docking domains (DDD), which bind to each other andallow the assembly of complex structures, ranging from dimers, trimers,tetramers, quintamers and hexamers. These form stable complexes in highyield without requirement for extensive purification. The DNL techniqueallows the assembly of monospecific, bispecific or multispecificantibodies. Any of the techniques known in the art for making bispecificor multispecific antibodies may be utilized in the practice of thepresently claimed methods.

In various embodiments, a conjugate as disclosed herein may be part of acomposite, multispecific antibody. Such antibodies may contain two ormore different antigen binding sites, with differing specificities. Themultispecific composite may bind to different epitopes of the sameantigen, or alternatively may bind to two different antigens.

DOCK-AND-LOCK® (DNL®)

In preferred embodiments, a bivalent or multivalent antibody is formedas a DOCK-AND-LOCK® (DNL®) complex (see, e.g., U.S. Pat. Nos. 7,521,056;7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070; 7,871,622;7,906,121; 7,906,118; 8,163,291; 7,901,680; 7,981,398; 8,003,111 and8,034,352, the Examples section of each of which is incorporated hereinby reference.) Generally, the technique takes advantage of the specificand high-affinity binding interactions that occur between a dimerizationand docking domain (DDD) sequence of the regulatory (R) subunits ofcAMP-dependent protein kinase (PKA) and an anchor domain (AD) sequencederived from any of a variety of AKAP proteins (Baillie et al., FEBSLetters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol.2004; 5: 959). The DDD and AD peptides may be attached to any protein,peptide or other molecule. Because the DDD sequences spontaneouslydimerize and bind to the AD sequence, the technique allows the formationof complexes between any selected molecules that may be attached to DDDor AD sequences.

Although the standard DNL® complex comprises a trimer with twoDDD-linked molecules attached to one AD-linked molecule, variations incomplex structure allow the formation of dimers, trimers, tetramers,pentamers, hexamers and other multimers. In some embodiments, the DNL®complex may comprise two or more antibodies, antibody fragments orfusion proteins which bind to the same antigenic determinant or to twoor more different antigens. The DNL® complex may also comprise one ormore other effectors, such as proteins, peptides, immunomodulators,cytokines, interleukins, interferons, binding proteins, peptide ligands,carrier proteins, toxins, ribonucleases such as onconase, inhibitoryoligonucleotides such as siRNA, antigens or xenoantigens, polymers suchas PEG, enzymes, therapeutic agents, hormones, cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents or any other molecule oraggregate.

PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has α and β isoforms (Scott, Pharmacol. Ther. 1991; 50:123). Thus,the four isoforms of PKA regulatory subunits are RIα, RIβ, RIIα andRIIβ. The R subunits have been isolated only as stable dimers and thedimerization domain has been shown to consist of the first 44amino-terminal residues of RIIα (Newlon et al., Nat. Struct. Biol. 1999;6:222). As discussed below, similar portions of the amino acid sequencesof other regulatory subunits are involved in dimerization and docking,each located near the N-terminal end of the regulatory subunit. Bindingof cAMP to the R subunits leads to the release of active catalyticsubunits for a broad spectrum of serine/threonine kinase activities,which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIa, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunits and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a DNL®complex through the introduction of cysteine residues into both the DDDand AD at strategic positions to facilitate the formation of disulfidebonds. The general methodology of the approach is as follows. Entity Ais constructed by linking a DDD sequence to a precursor of A, resultingin a first component hereafter referred to as a. Because the DDDsequence would effect the spontaneous formation of a dimer, A would thusbe composed of az. Entity B is constructed by linking an AD sequence toa precursor of B, resulting in a second component hereafter referred toas b. The dimeric motif of DDD contained in a₂ will create a dockingsite for binding to the AD sequence contained in b, thus facilitating aready association of a₂ and b to form a binary, trimeric complexcomposed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsshould bring the reactive thiol groups placed onto both the DDD and ADinto proximity (Chmura et al., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various combinations oflinkers, adaptor modules and precursors, a wide variety of DNL®constructs of different stoichiometry may be produced and used (see,e.g., U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNL °construct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

In various embodiments, an antibody or antibody fragment may beincorporated into a DNL® complex by, for example, attaching a DDD or ADmoiety to the C-terminal end of the antibody heavy chain, as describedin detail below. In more preferred embodiments, the DDD or AD moiety,more preferably the AD moiety, may be attached to the C-terminal end ofthe antibody light chain (see, e.g., U.S. patent application Ser. No.13/901,737, filed May 24, 2013, the Examples section of which isincorporated herein by reference.)

For different types of DNL® constructs, different AD or DDD sequencesmay be utilized. Exemplary DDD and AD sequences are provided below.

DDD1 (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 3) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 4)CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 are based on the DDDsequence of the human RIIα isoform of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human Ma form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 5) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAKDDD3C (SEQ ID NO: 6) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 7) CGFEELAWKIAKMIWSDVFQQGC

In other alternative embodiments, other sequence variants of AD and/orDDD moieties may be utilized in construction of the DNL® complexes. Forexample, there are only four variants of human PKA DDD sequences,corresponding to the DDD moieties of PKA RIα, RIIα, RIβ and RIIβ. TheRIIα DDD sequence is the basis of DDD1 and DDD2 disclosed above. Thefour human PKA DDD sequences are shown below. The DDD sequencerepresents residues 1-44 of RIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66of RIβ. (Note that the sequence of DDD1 is modified slightly from thehuman PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 8)SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEA K PKA RIβ(SEQ ID NO: 9) SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENR QILAPKA RIIα (SEQ ID NO: 10) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQPKA RIIβ (SEQ ID NO: 11) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Id.). It has beenreported that G1m1 antibodies contain allotypic sequences that tend toinduce an immune response when administered to non-G1m1 (nG1m1)recipients, such as G1m3 patients (Id.). Non-G1m1 allotype antibodiesare not as immunogenic when administered to G1m1 patients (Id.).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1m1 and nG1m1 allotype antibodies is shown for theexemplary antibodies rituximab (SEQ ID NO:12) and veltuzumab (SEQ IDNO:13).

Rituximab heavy chain variable region sequence (SEQ ID NO: 12)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Veltuzumab heavy chain variable region(SEQ ID NO: 13) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The G1m1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotypoe characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies. Table 3compares the allotype sequences of rituximab vs. veltuzumab. As shown inTable 3, rituximab (G1m17,1) is a DEL allotype IgG1, with an additionalsequence variation at Kabat position 214 (heavy chain CH1) of lysine inrituximab vs. arginine in veltuzumab. It has been reported thatveltuzumab is less immunogenic in subjects than rituximab (see, e.g.,Morchhauser et al., 2009, J Clin Oncol 27:3346-53; Goldenberg et al.,2009, Blood 113:1062-70; Robak & Robak, 2011, BioDrugs 25:13-25), aneffect that has been attributed to the difference between humanized andchimeric antibodies. However, the difference in allotypes between theEEM and DEL allotypes likely also accounts for the lower immunogenicityof veltuzumab.

TABLE 3 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes Complete (allo- 356/ (allo- (allo- allotype 214type) 358 type) 431 type) Rituximab G1m17,1 K 17 D/L 1 A — VeltuzumabG1m3 R 3 E/M — A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of G1m3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. The skilled artisan will be aware that,in general, amino acid substitutions typically involve the replacementof an amino acid with another amino acid of relatively similarproperties (i.e., conservative amino acid substitutions). The propertiesof the various amino acids and effect of amino acid substitution onprotein structure and function have been the subject of extensive studyand knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Antibody-Drug Conjugates

In certain embodiments, a cytotoxic drug or other therapeutic ordiagnostic agent may be covalently attached to an antibody or antibodyfragment to form an ADC. In some embodiments, a drug or other agent maybe attached to an antibody or fragment thereof via a carrier moiety.Carrier moieties may be attached, for example to reduced SH groupsand/or to carbohydrate side chains. A carrier moiety can be attached atthe hinge region of a reduced antibody component via disulfide bondformation. Alternatively, such agents can be attached using aheterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the carrier moiety canbe conjugated via a carbohydrate moiety in the Fc region of theantibody.

Methods for conjugating functional groups to antibodies via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, the Examples section of which is incorporated herein byreference. The general method involves reacting an antibody having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody component of the ADC is anantibody fragment. However, it is possible to introduce a carbohydratemoiety into the light chain variable region of a full length antibody orantibody fragment. See, for example, Leung et al., J. Immunol. 154: 5919(1995); U.S. Pat. Nos. 5,443,953 and 6,254,868, the Examples section ofwhich is incorporated herein by reference. The engineered carbohydratemoiety is used to attach the therapeutic or diagnostic agent.

An alternative method for attaching carrier moieties to a targetingmolecule involves use of click chemistry reactions. The click chemistryapproach was originally conceived as a method to rapidly generatecomplex substances by joining small subunits together in a modularfashion. (See, e.g., Kolb et al., 2004, Angew Chem Int Ed 40:3004-31;Evans, 2007, Aust J Chem 60:384-95.) Various forms of click chemistryreaction are known in the art, such as the Huisgen 1,3-dipolarcycloaddition copper catalyzed reaction (Tornoe et al., 2002, J OrganicChem 67:3057-64), which is often referred to as the “click reaction.”Other alternatives include cycloaddition reactions such as theDiels-Alder, nucleophilic substitution reactions (especially to smallstrained rings like epoxy and aziridine compounds), carbonyl chemistryformation of urea compounds and reactions involving carbon-carbon doublebonds, such as alkynes in thiol-yne reactions.

The azide alkyne Huisgen cycloaddition reaction uses a copper catalystin the presence of a reducing agent to catalyze the reaction of aterminal alkyne group attached to a first molecule. In the presence of asecond molecule comprising an azide moiety, the azide reacts with theactivated alkyne to form a 1,4-disubstituted 1,2,3-triazole. The coppercatalyzed reaction occurs at room temperature and is sufficientlyspecific that purification of the reaction product is often notrequired. (Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tornoe etal., 2002, J Org Chem 67:3057.) The azide and alkyne functional groupsare largely inert towards biomolecules in aqueous medium, allowing thereaction to occur in complex solutions. The triazole formed ischemically stable and is not subject to enzymatic cleavage, making theclick chemistry product highly stable in biological systems. Althoughthe copper catalyst is toxic to living cells, the copper-based clickchemistry reaction may be used in vitro for ADC formation.

A copper-free click reaction has been proposed for covalent modificationof biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc126:15046-47.) The copper-free reaction uses ring strain in place of thecopper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction(Id.) For example, cyclooctyne is an 8-carbon ring structure comprisingan internal alkyne bond. The closed ring structure induces a substantialbond angle deformation of the acetylene, which is highly reactive withazide groups to form a triazole. Thus, cyclooctyne derivatives may beused for copper-free click reactions (Id.)

Another type of copper-free click reaction was reported by Ning et al.(2010, Angew Chem Int Ed 49:3065-68), involving strain-promotedalkyne-nitrone cycloaddition. To address the slow rate of the originalcyclooctyne reaction, electron-withdrawing groups are attached adjacentto the triple bond (Id.) Examples of such substituted cyclooctynesinclude difluorinated cyclooctynes, 4-dibenzocyclooctynol andazacyclooctyne (Id.) An alternative copper-free reaction involvedstrain-promoted alkyne-nitrone cycloaddition to give N-alkylatedisoxazolines (Id.) The reaction was reported to have exceptionally fastreaction kinetics and was used in a one-pot three-step protocol forsite-specific modification of peptides and proteins (Id.) Nitrones wereprepared by the condensation of appropriate aldehydes withN-methylhydroxylamine and the cycloaddition reaction took place in amixture of acetonitrile and water (Id.) These and other known clickchemistry reactions may be used to attach carrier moieties to antibodiesin vitro.

Agard et al. (2004, J Am Chem Soc 126:15046-47) demonstrated that arecombinant glycoprotein expressed in CHO cells in the presence ofperacetylated N-azidoacetylmannosamine resulted in the bioincorporationof the corresponding N-azidoacetyl sialic acid in the carbohydrates ofthe glycoprotein. The azido-derivatized glycoprotein reactedspecifically with a biotinylated cyclooctyne to form a biotinylatedglycoprotein, while control glycoprotein without the azido moietyremained unlabeled (Id.) Laughlin et al. (2008, Science 320:664-667)used a similar technique to metabolically label cell-surface glycans inzebrafish embryos incubated with peracetylatedN-azidoacetylgalactosamine. The azido-derivatized glycans reacted withdifluorinated cyclooctyne (DIFO) reagents to allow visualization ofglycans in vivo.

The Diels-Alder reaction has also been used for in vivo labeling ofmolecules. Rossin et al. (2010, Angew Chem Int Ed 49:3375-78) reported a52% yield in vivo between a tumor-localized anti-TAG72 (CC49) antibodycarrying a trans-cyclooctene (TCO) reactive moiety and an ¹¹¹In-labeledtetrazine DOTA derivative. The TCO-labeled CC49 antibody wasadministered to mice bearing colon cancer xenografts, followed 1 daylater by injection of ¹¹¹In-labeled tetrazine probe (Id.) The reactionof radiolabeled probe with tumor localized antibody resulted inpronounced radioactivity localization in the tumor, as demonstrated bySPECT imaging of live mice three hours after injection of radiolabeledprobe, with a tumor-to-muscle ratio of 13:1 (Id.) The results confirmedthe in vivo chemical reaction of the TCO and tetrazine-labeledmolecules.

Modifications of click chemistry reactions are suitable for use in vitroor in vivo. Reactive targeting molecule may be formed either by eitherchemical conjugation or by biological incorporation. The targetingmolecule, such as an antibody or antibody fragment, may be activatedwith an azido moiety, a substituted cyclooctyne or alkyne group, or anitrone moiety. Where the targeting molecule comprises an azido ornitrone group, the corresponding targetable construct will comprise asubstituted cyclooctyne or alkyne group, and vice versa. Such activatedmolecules may be made by metabolic incorporation in living cells, asdiscussed above.

Alternatively, methods of chemical conjugation of such moieties tobiomolecules are well known in the art, and any such known method may beutilized. General methods of ADC formation are disclosed, for example,in U.S. Pat. Nos. 4,699,784; 4,824,659; 5,525,338; 5,677,427; 5,697,902;5,716,595; 6,071,490; 6,187,284; 6,306,393; 6,548,275; 6,653,104;6,962,702; 7,033,572; 7,147,856; and 7,259,240, the Examples section ofeach incorporated herein by reference.

The preferred conjugation protocol is based on a thiol-maleimide, athiol-vinylsulfone, a thiol-bromoacetamide, or a thiol-iodoacetamidereaction that is facile at neutral or acidic pH. This obviates the needfor higher pH conditions for conjugations as, for instance, would benecessitated when using active esters. Further details of exemplaryconjugation protocols are described below in the Examples section.

Therapeutic Treatment

In another aspect, the invention relates to a method of treating asubject, comprising administering to a subject a therapeuticallyeffective amount of an antibody-drug conjugate (ADC) and an ABCG2inhibitor as described herein. Diseases that may be treated with theADCs and ABCG2 inhibitors include, but are not limited to B-cellmalignancies (e.g., non-Hodgkin's lymphoma, mantle cell lymphoma,multiple myeloma, Hodgkin's lymphoma, diffuse large B cell lymphoma,Burkitt lymphoma, follicular lymphoma, acute lymphocytic leukemia,chronic lymphocytic leukemia, hairy cell leukemia) using, for example ananti-CD22 antibody such as the hLL2 MAb (epratuzumab, see U.S. Pat. No.6,183,744), against another CD22 epitope (hRFB4) or antibodies againstother B cell antigens, such as CD19, CD20, CD21, CD22, CD23, CD37, CD40,CD40L, CD52, CD74, CD80 or HLA-DR. Other diseases include, but are notlimited to, adenocarcinomas of endodermally-derived digestive systemepithelia, cancers such as breast cancer and non-small cell lung cancer,and other carcinomas, sarcomas, glial tumors, myeloid leukemias, etc. Inparticular, antibodies against an antigen, e.g., an oncofetal antigen,produced by or associated with a malignant solid tumor or hematopoieticneoplasm, e.g., a gastrointestinal, stomach, colon, esophageal, liver,lung, breast, pancreatic, liver, prostate, ovarian, testicular, brain,bone or lymphatic tumor, a sarcoma or a melanoma, are advantageouslyused. Such therapeutics can be given once or repeatedly, depending onthe disease state and tolerability of the conjugate, and can also beused optionally in combination with other therapeutic modalities, suchas surgery, external radiation, radioimmunotherapy, immunotherapy,chemotherapy, antisense therapy, interference RNA therapy, gene therapy,and the like. Each combination will be adapted to the tumor type, stage,patient condition and prior therapy, and other factors considered by themanaging physician.

As used herein, the term “subject” refers to any animal (i.e.,vertebrates and invertebrates) including, but not limited to mammals,including humans. It is not intended that the term be limited to aparticular age or sex. Thus, adult and newborn subjects, as well asfetuses, whether male or female, are encompassed by the term. Dosesgiven herein are for humans, but can be adjusted to the size of othermammals, as well as children, in accordance with weight or square metersize.

In a preferred embodiment, an ADC comprising an anti-Trop-2 antibodysuch as the hRS7 MAb can be used to treat carcinomas such as carcinomasof the esophagus, pancreas, lung, stomach, colon and rectum, urinarybladder, breast, ovary, uterus, kidney and prostate, as disclosed inU.S. Pat. Nos. 7,238,785; 7,517,964 and 8,084,583, the Examples sectionof which is incorporated herein by reference. An hRS7 antibody is ahumanized antibody that comprises light chaincomplementarity-determining region (CDR) sequences CDR1 (KASQDVSIAVA,SEQ ID NO:14); CDR2 (SASYRYT, SEQ ID NO:15); and CDR3 (QQHYITPLT, SEQ IDNO:16) and heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:17); CDR2(WINTYTGEPTYTDDFKG, SEQ ID NO:18) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:19)

In another preferred embodiment, an ADC comprising an anti-CEACAM5antibody (e.g., hMN-14, labretuzumab) and/or an anti-CEACAM6 antibodymay be used to treat any of a variety of cancers that express CEACAM5and/or CEACAM6, as disclosed in U.S. Pat. Nos. 7,541,440; 7,951,369;5,874,540; 6,676,924 and 8,267,865, the Examples section of eachincorporated herein by reference. Solid tumors that may be treated usinganti-CEACAM5, anti-CEACAM6, or a combination of the two include but arenot limited to breast, lung, pancreatic, esophageal, medullary thyroid,ovarian, colon, rectum, urinary bladder, mouth and stomach cancers. Amajority of carcinomas, including gastrointestinal, respiratory,genitourinary and breast cancers express CEACAM5 and may be treated withthe subject ADCs. An hMN-14 antibody is a humanized antibody thatcomprises light chain variable region CDR sequences CDR1 (KASQDVGTSVA;SEQ ID NO:20), CDR2 (WTSTRHT; SEQ ID NO:21), and CDR3 (QQYSLYRS; SEQ IDNO:22), and the heavy chain variable region CDR sequences CDR1 (TYWMS;SEQ ID NO:23), CDR2 (EIHPDSSTINYAPSLKD; SEQ ID NO:24) and CDR3(LYFGFPWFAY; SEQ ID NO:25).

In another preferred embodiment, an ADC comprising an anti-CD22 antibody(e.g., hLL2, epratuzumab, disclosed in U.S. Pat. Nos. 5,789,554;6,183,744; 6,187,287; 6,306,393; 7,074,403 and 7,641,901, the Examplessection of each incorporated herein by reference, or the chimeric orhumanized RFB4 antibody) may be used to treat any of a variety ofcancers that express CD22, including but not limited to indolent formsof B-cell lymphomas, aggressive forms of B-cell lymphomas, chroniclymphatic leukemias, acute lymphatic leukemias, non-Hodgkin's lymphoma,Hodgkin's lymphoma, Burkitt lymphoma, follicular lymphoma or diffuseB-cell lymphoma. An hLL2 antibody is a humanized antibody comprisinglight chain CDR sequences CDR1 (KSSQSVLYSANHKYLA, SEQ ID NO:26), CDR2(WASTRES, SEQ ID NO:27), and CDR3 (HQYLSSWTF, SEQ ID NO:28) and theheavy chain CDR sequences CDR1 (SYWLH, SEQ ID NO:29), CDR2(YINPRNDYTEYNQNFKD, SEQ ID NO:30), and CDR3 (RDITTFY, SEQ ID NO:31)

In another preferred embodiment, an ADC comprising an anti-HLA-DR MAb,such as hL243, can be used to treat lymphoma, leukemia, cancers of theskin, esophagus, stomach, colon, rectum, pancreas, lung, breast, ovary,bladder, endometrium, cervix, testes, kidney, liver, melanoma or otherHLA-DR-producing tumors, as disclosed in U.S. Pat. No. 7,612,180, theExamples section of which is incorporated herein by reference. An hL243antibody is a humanized antibody comprising the heavy chain CDRsequences CDR1 (NYGMN, SEQ ID NO:32), CDR2 (WINTYTREPTYADDFKG, SEQ IDNO:33), and CDR3 (DITAVVPTGFDY, SEQ ID NO:34) and light chain CDRsequences CDR1 (RASENIYSNLA, SEQ ID NO:35), CDR2 (AASNLAD, SEQ IDNO:36), and CDR3 (QHFWTTPWA, SEQ ID NO:37).

In another preferred embodiment, therapeutic conjugates comprising ananti-CD74 antibody (e.g., hLL1, milatuzumab, disclosed in U.S. Pat. Nos.7,074,403; 7,312,318; 7,772,373; 7,919,087 and 7,931,903, the Examplessection of each incorporated herein by reference) may be used to treatany of a variety of cancers that express CD74, including but not limitedto renal, lung, intestinal, stomach, breast, prostate or ovarian cancer,as well as several hematological cancers, such as multiple myeloma,chronic lymphocytic leukemia, acute lymphoblastic leukemia, non-Hodgkinlymphoma, and Hodgkin lymphoma. An hLL1 antibody is a humanized antibodycomprising the light chain CDR sequences CDR1 (RSSQSLVHRNGNTYLH; SEQ IDNO:38), CDR2 (TVSNRFS; SEQ ID NO:39), and CDR3 (SQSSHVPPT; SEQ ID NO:40)and the heavy chain variable region CDR sequences CDR1 (NYGVN; SEQ IDNO:41), CDR2 (WINPNTGEPTFDDDFKG; SEQ ID NO:42), and CDR3 (SRGKNEAWFAY;SEQ ID NO:43).

In another preferred embodiment, therapeutic conjugates comprising ananti-CD20 MAb, such as veltuzumab (hA20), 1F5, obinutuzumab (GA101), orrituximab, can be used to treat lymphoma, leukemia, immunethrombocytopenic purpura, systemic lupus erythematosus, Sjogren'ssyndrome, Evans syndrome, arthritis, arteritis, pemphigus vulgaris,renal graft rejection, cardiac graft rejection, rheumatoid arthritis,Burkitt lymphoma, non-Hodgkin's lymphoma, follicular lymphoma, smalllymphocytic lymphoma, diffuse B-cell lymphoma, marginal zone lymphoma,chronic lymphocytic leukemia, acute lymphocytic leukemia, Type Idiabetes mellitus, GVHD, multiple sclerosis or multiple myeloma, asdisclosed in U.S. Pat. No. 7,435,803 or 8,287,864, the Examples sectionof each incorporated herein by reference. An hA20 (veltuzumab) antibodyis a humanized antibody comprising the light chain CDR sequences CDRL1(RASSSVSYIH, SEQ ID NO:44), CDRL2 (ATSNLAS, SEQ ID NO:45) and CDRL3(QQWTSNPPT, SEQ ID NO:46) and heavy chain CDR sequences CDRH1 (SYNMH,SEQ ID NO:47), CDRH2 (AIYPGNGDTSYNQKFKG, SEQ ID NO:48) and CDRH3(STYYGGDWYFDV, SEQ ID NO:49).

In another preferred embodiment, therapeutic conjugates comprising ananti-MUC-5ac antibody, such as an hPAM4 antibody, can be used to treattumors that express MUC-5ac, such as pancreatic cancer and colorectalcancer. An hPAM4 is a humanized antibody comprising the light chainvariable region complementarity-determining region (CDR) sequences CDR1(SASSSVSSSYLY, SEQ ID NO:50); CDR2 (STSNLAS, SEQ ID NO:51); and CDR3(HQWNRYPYT, SEQ ID NO:52); and the heavy chain CDR sequences CDR1(SYVLH, SEQ ID NO:53); CDR2 (YINPYNDGTQYNEKFKG, SEQ ID NO:54) and CDR3(GFGGSYGFAY, SEQ ID NO:55)

In a preferred embodiment, the antibodies that are used in the treatmentof human disease are human or humanized (CDR-grafted) versions ofantibodies; although murine and chimeric versions of antibodies can beused. Same species IgG molecules as delivery agents are mostly preferredto minimize immune responses. This is particularly important whenconsidering repeat treatments. For humans, a human or humanized IgGantibody is less likely to generate an anti-IgG immune response frompatients. Antibodies such as hLL1 and hLL2 rapidly internalize afterbinding to internalizing antigen on target cells, which means that thechemotherapeutic drug being carried is rapidly internalized into cellsas well. However, antibodies that have slower rates of internalizationcan also be used to effect selective therapy.

In a preferred embodiment, a more effective incorporation into cells canbe accomplished by using multivalent, multispecific or multivalent,monospecific antibodies. Examples of such bivalent and bispecificantibodies are found in U.S. Pat. Nos. 7,387,772; 7,300,655; 7,238,785;and 7,282,567, the Examples section of each of which is incorporatedherein by reference. These multivalent or multispecific antibodies areparticularly preferred in the targeting of cancers, which expressmultiple antigen targets and even multiple epitopes of the same antigentarget, but which often evade antibody targeting and sufficient bindingfor immunotherapy because of insufficient expression or availability ofa single antigen target on the cell. By targeting multiple antigens orepitopes, said antibodies show a higher binding and residence time onthe target, thus affording a higher saturation with the drug beingtargeted in this invention.

In another preferred embodiment, the ADCs can be used to treatautoimmune disease or immune system dysfunction (e.g., graft-versus-hostdisease, organ transplant rejection). Antibodies of use to treatautoimmune/immune dysfunction disease may bind to exemplary antigensincluding, but not limited to, BCL-1, BCL-2, BCL-6, CD1a, CD2, CD3, CD4,CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20,CD21, CD22, CD23, CD25, CD33, CD34, CD38, CD40, CD40L, CD41a, CD43,CD45, CD55, CD56, CCD57, CD59, CD64, CD71, CD74, CD79a, CD79b, CD117,CD138, FMC-7 and HLA-DR. Antibodies that bind to these and other targetantigens, discussed above, may be used to treat autoimmune or immunedysfunction diseases. Autoimmune diseases that may be treated with ADCsmay include acute idiopathic thrombocytopenic purpura, chronicidiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea,myasthenia gravis, systemic lupus erythematosus, lupus nephritis,rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetesmellitus, Henoch-Schonlein purpura, post-streptococcal nephritis,erythema nodosum, Takayasu's arteritis, ANCA-associated vasculitides,Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis obliterans, Sjogren's syndrome, primary biliarycirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronicactive hepatitis, polymyositis/dermatomyositis, polychondritis, bullouspemphigoid, pemphigus vulgaris, Wegener's granulomatosis, membranousnephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

In various embodiments, the anti-Trop-2 ADC and ABCG2 inhibitor may beused in further combination with one or more chemotherapeutic drugs suchas vinca alkaloids, anthracyclines, epidophyllotoxins, taxanes,antimetabolites, tyrosine kinase inhibitors, alkylating agents,antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic andproapoptotic agents, particularly doxorubicin, methotrexate, taxol,other camptothecins, and others from these and other classes ofanticancer agents, and the like. Other cancer chemotherapeutic drugsinclude nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes,folic acid analogs, pyrimidine analogs, purine analogs, platinumcoordination complexes, hormones, and the like. Suitablechemotherapeutic agents are described in REMINGTON'S PHARMACEUTICALSCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN ANDGILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillanPublishing Co. 1985), as well as revised editions of these publications.Other suitable chemotherapeutic agents, such as experimental drugs, areknown to those of skill in the art.

Exemplary drugs of use include, but are not limited to, 5-fluorouracil,afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib,AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib,bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin,cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine,dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin,daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicinglucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib,entinostat, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, exemestane, fingolimod,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, farnesyl-protein transferase inhibitors, flavopiridol,fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine,hydroxyurea, ibrutinib, idarubicin, idelali sib, ifosfamide, imatinib,L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine,vinblastine, vincristine, vinca alkaloids and ZD1839. Such agents may bepart of the conjugates described herein or may alternatively beadministered in combination with the described conjugates, either priorto, simultaneously with or after the conjugate.

Yet another class of therapeutic agent that may be used in addition tothe combination of ADC and ABCG2 inhibitor may comprise one or moreimmunomodulators. Immunomodulators of use may be selected from acytokine, a stem cell growth factor, a lymphotoxin, an hematopoieticfactor, a colony stimulating factor (CSF), an interferon (IFN),erythropoietin, thrombopoietin and a combination thereof. Specificallyuseful are lymphotoxins such as tumor necrosis factor (TNF),hematopoietic factors, such as interleukin (IL), colony stimulatingfactor, such as granulocyte-colony stimulating factor (G-CSF) orgranulocyte macrophage-colony stimulating factor (GM-CSF), interferon,such as interferons-α, -β, -γ or -λ, and stem cell growth factor, suchas that designated “S1 factor”. Included among the cytokines are growthhormones such as human growth hormone, N-methionyl human growth hormone,and bovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand lymphotoxin (LT). As used herein, the term cytokine includesproteins from natural sources or from recombinant cell culture andbiologically active equivalents of the native sequence cytokines.

Chemokines of use include RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.

An alternative therapeutic agent of use is a tyrosine kinase inhibitor.A variety of tyrosine kinase inhibitors are known in the art and anysuch known therapeutic agent may be utilized. Exemplary tyrosine kinaseinhibitors include, but are not limited to canertinib, dasatinib,erlotinib, gefitinib, imatinib, lapatinib, leflunomide, nilotinib,pazopanib, semaxinib, sorafenib, sunitinib, sutent and vatalanib. Aspecific class of tyrosine kinase inhibitor is the Bruton tyrosinekinase inhibitor. Bruton tyrosine kinase (Btk) has a well-defined rolein B-cell development. Bruton kinase inhibitors include, but are notlimited to, PCI-32765 (ibrutinib), PCI-45292, GDC-0834, LFM-A13 andRN486.

The person of ordinary skill will realize that the subject ADC and ABCG2inhibitor, may be used alone or in combination with one or more othertherapeutic agents, such as a second antibody, second antibody fragment,second ADC, radionuclide, toxin, drug, chemotherapeutic agent, radiationtherapy, chemokine, cytokine, immunomodulator, enzyme, hormone,oligonucleotide, RNAi or siRNA. Such additional therapeutic agents maybe administered separately, in combination with, or attached to thesubject ADC.

Formulation and Administration

Suitable routes of administration of the conjugates include, withoutlimitation, oral, parenteral, subcutaneous, rectal, transmucosal,intestinal administration, intramuscular, intramedullary, intrathecal,direct intraventricular, intravenous, intravitreal, intraperitoneal,intranasal, or intraocular injections. The preferred routes ofadministration are parenteral. Alternatively, one may administer thecompound in a local rather than systemic manner, for example, viainjection of the compound directly into a solid tumor.

ADCs and ABCG2 inhibitors can be formulated according to known methodsto prepare pharmaceutically useful compositions, whereby the ADC and/orABCG2 inhibitor is combined in a mixture with a pharmaceuticallysuitable excipient. Sterile phosphate-buffered saline is one example ofa pharmaceutically suitable excipient. Other suitable excipients arewell-known to those in the art. See, for example, Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

In a preferred embodiment, the ADC is formulated in Good's biologicalbuffer (pH 6-7), using a buffer selected from the group consisting ofN-(2-acetamido)-2-aminoethanesulfonic acid (ACES);N-(2-acetamido)iminodiacetic acid (ADA);N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES);4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES);2-(N-morpholino)ethanesulfonic acid (IVIES);3-(N-morpholino)propanesulfonic acid (MOPS);3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO); andpiperazine-N,N′-bis(2-ethanesulfonic acid) [Pipes]. More preferredbuffers are MES or MOPS, preferably in the concentration range of 20 to100 mM, more preferably about 25 mM. Most preferred is 25 mM MES, pH6.5. The formulation may further comprise 25 mM trehalose and 0.01% v/vpolysorbate 80 as excipients, with the final buffer concentrationmodified to 22.25 mM as a result of added excipients. The preferredmethod of storage is as a lyophilized formulation of the conjugates,stored in the temperature range of −20° C. to 2° C., with the mostpreferred storage at 2° C. to 8° C.

The ADC can be formulated for intravenous administration via, forexample, bolus injection, slow infusion or continuous infusion.Preferably, the antibody of the present invention is infused over aperiod of less than about 4 hours, and more preferably, over a period ofless than about 3 hours. For example, the first 25-50 mg could beinfused within 30 minutes, preferably even 15 min, and the remainderinfused over the next 2-3 hrs. Formulations for injection can bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

ABCG2 inhibitors may be prepared in a capsule or tablet suitable fororal administration, or may also be administered intravenously,subcutaneously, or by other parenteral administration.

Additional pharmaceutical methods may be employed to control theduration of action of the ADC and/or ABCG2 inhibitor. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the active agents. For example, biocompatible polymers includematrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10:1446 (1992). The rate of release ofan agent from such a matrix depends upon the molecular weight of theagent, the amount of agent within the matrix, and the size of dispersedparticles. Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et al.,supra. Other solid dosage forms are described in Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

Generally, the dosage of an administered ADC and/or ABCG2 inhibitor forhumans will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. It may be desirable to provide the recipient with a dosage ofADC that is in the range of from about 1 mg/kg to 24 mg/kg as a singleintravenous infusion, although a lower or higher dosage also may beadministered as circumstances dictate. A dosage of 1-20 mg/kg for a 70kg patient, for example, is 70-1,400 mg, or 41-824 mg/m² for a 1.7-mpatient. The dosage may be repeated as needed, for example, once perweek for 4-10 weeks, once per week for 8 weeks, or once per week for 4weeks. It may also be given less frequently, such as every other weekfor several months, or monthly or quarterly for many months, as neededin a maintenance therapy. Preferred dosages may include, but are notlimited to, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 22mg/kg and 24 mg/kg. Any amount in the range of 1 to 24 mg/kg may beused. The dosage is preferably administered multiple times, once ortwice a week. A minimum dosage schedule of 4 weeks, more preferably 8weeks, more preferably 16 weeks or longer may be used. The schedule ofadministration may comprise administration once or twice a week, on acycle selected from the group consisting of: (i) weekly; (ii) everyother week; (iii) one week of therapy followed by two, three or fourweeks off; (iv) two weeks of therapy followed by one, two, three or fourweeks off; (v) three weeks of therapy followed by one, two, three, fouror five week off; (vi) four weeks of therapy followed by one, two,three, four or five week off; (vii) five weeks of therapy followed byone, two, three, four or five week off; and (viii) monthly. The cyclemay be repeated 4, 6, 8, 10, 12, 16 or 20 times or more.

Alternatively, an ADC and/or ABCG2 inhibitor may be administered as onedosage every 2 or 3 weeks, repeated for a total of at least 3 dosages.Or, twice per week for 4-6 weeks. If the dosage is lowered toapproximately 200-300 mg/m² (340 mg per dosage for a 1.7-m patient, or4.9 mg/kg for a 70 kg patient), it may be administered once or eventwice weekly for 4 to 10 weeks. Alternatively, the dosage schedule maybe decreased, namely every 2 or 3 weeks for 2-3 months. It has beendetermined, however, that even higher doses, such as 12 mg/kg onceweekly or once every 2-3 weeks can be administered by slow i.v.infusion, for repeated dosing cycles. The dosing schedule can optionallybe repeated at other intervals and dosage may be given through variousparenteral routes, with appropriate adjustment of the dose and schedule.

In preferred embodiments, the ADCs and ABCG2 inhibitors are of use fortherapy of cancer. Examples of cancers include, but are not limited to,carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia,myeloma, or lymphoid malignancies. More particular examples of suchcancers are noted below and include: squamous cell cancer (e.g.,epithelial squamous cell cancer), Ewing sarcoma, Wilms tumor,astrocytomas, lung cancer including small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung and squamous carcinoma ofthe lung, cancer of the peritoneum, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma multiforme,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,hepatocellular carcinoma, neuroendocrine tumors, medullary thyroidcancer, differentiated thyroid carcinoma, breast cancer, ovarian cancer,colon cancer, rectal cancer, endometrial cancer or uterine carcinoma,salivary gland carcinoma, kidney or renal cancer, prostate cancer,vulvar cancer, anal carcinoma, penile carcinoma, as well ashead-and-neck cancer. The term “cancer” includes primary malignant cellsor tumors (e.g., those whose cells have not migrated to sites in thesubject's body other than the site of the original malignancy or tumor)and secondary malignant cells or tumors (e.g., those arising frommetastasis, the migration of malignant cells or tumor cells to secondarysites that are different from the site of the original tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias; e.g., acute lymphocytic leukemia, acute myelocytic leukemia[including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia]) and chronic leukemias (e.g., chronic myelocytic[granulocytic] leukemia and chronic lymphocytic leukemia), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Autoimmune diseases that may be treated with ADCs may include acute andchronic immune thrombocytopenias, dermatomyositis, Sydenham's chorea,myasthenia gravis, systemic lupus erythematosus, lupus nephritis,rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetesmellitus, Henoch-Schonlein purpura, post-streptococcal nephritis,erythema nodosum, Takayasu's arteritis, ANCA-associated vasculitides,Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis obliterans, Sjogren's syndrome, primary biliarycirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronicactive hepatitis, polymyositis/dermatomyositis, polychondritis, bullouspemphigoid, pemphigus vulgaris, Wegener's granulomatosis, membranousnephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

Kits

Various embodiments may concern kits containing components suitable fortreating diseased tissue in a patient. Exemplary kits may contain atleast one ADC and/or ABCG2 inhibitor as described herein. If thecomposition containing components for administration is not formulatedfor delivery via the alimentary canal, such as by oral delivery, adevice capable of delivering the kit components through some other routemay be included. One type of device, for applications such as parenteraldelivery, is a syringe that is used to inject the composition into thebody of a subject. Inhalation devices may also be used.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES

Various embodiments of the present invention are illustrated by thefollowing examples, without limiting the scope thereof.

Example 1. Production and Use of Anti-Trop-2-SN-38 Antibody-DrugConjugate

The humanized RS7 (hRS7) anti-Trop-2 antibody was produced as describedin U.S. Pat. No. 7,238,785, the Figures and Examples section of whichare incorporated herein by reference. SN-38 attached to a CL2A linkerwas produced and conjugated to hRS7 (anti-Trop-2), hPAM4 (anti-MUC5ac),hA20 (anti-CD20) or hMN-14 (anti-CEACAM5) antibodies according to U.S.Pat. No. 7,999,083 (Example 10 and 12 of which are incorporated hereinby reference). The conjugation protocol resulted in a ratio of about 6SN-38 molecules attached per antibody molecule.

Immune-compromised athymic nude mice (female), bearing subcutaneoushuman pancreatic or colon tumor xenografts were treated with eitherspecific CL2A-SN-38 conjugate or control conjugate or were leftuntreated. The therapeutic efficacies of the specific conjugates wereobserved. FIG. 1 shows a Capan 1 pancreatic tumor model, whereinspecific CL2A-SN-38 conjugates of hRS7 (anti-Trop-2), hPAM4(anti-MUC-5ac), and hMN-14 (anti-CEACAM5) antibodies showed betterefficacies than control hA20-CL2A-SN-38 conjugate (anti-CD20) anduntreated control. Similarly in a BXPC3 model of human pancreaticcancer, the specific hRS7-CL2A-SN-38 showed better therapeutic efficacythan control treatments (FIG. 2).

Example 2. ADCC Activity of Anti-Trop-2 ADCs

The ADCC activity of various hRS7-ADC conjugates was determined incomparison to hRS7 IgG (FIG. 3). PBMCs were purified from bloodpurchased from the Blood Center of New Jersey. A Trop-2-positive humanpancreatic adenocarcinoma cell line (BxPC-3) was used as the target cellline with an effector to target ratio of 100:1. ADCC mediated by hRS7IgG was compared to hRS7-Pro-2-PDox, hRS7-CL2A-SN-38, and the reducedand capped hRS7-NEM. All were used at 33.3 nM.

Results are shown in FIG. 3. Overall activity was low, but significant.There was 8.5% specific lysis for the hRS7 IgG which was notsignificantly different from hRS7-Pro-2-PDox. Both were significantlybetter than hLL2 control and hRS7-NEM and hRS7-SN-38 (P<0.02, two-tailedt-test). There was no difference between hRS7-NEM and hRS7-SN-38.

Example 3. Efficacy of Anti-Trop-2-SN-38 ADC Against Diverse EpithelialCancers In Vivo

Abstract

The purpose of this study was to evaluate the efficacy of anSN-38-anti-Trop-2 (hRS7) ADC against several human solid tumor types,and to assess its tolerability in mice and monkeys, the latter withtissue cross-reactivity to hRS7 similar to humans. Two SN-38derivatives, CL2-SN-38 and CL2A-SN-38, were conjugated to theanti-Trop-2-humanized antibody, hRS7. The ADCs were characterized invitro for stability, binding, and cytotoxicity. Efficacy was tested infive different human solid tumor-xenograft models that expressed Trop-2antigen. Toxicity was assessed in mice and in Cynomolgus monkeys.

The hRS7 conjugates of the two SN-38 derivatives were equivalent in drugsubstitution (−6), cell binding (K_(d)˜1.2 nmol/L), cytotoxicity(IC₅₀˜2.2 nmol/L), and serum stability in vitro (t/_(1/2)˜20 hours).Exposure of cells to the ADC demonstrated signaling pathways leading toPARP cleavage, but differences versus free SN-38 in p53 and p21upregulation were noted. Significant antitumor effects were produced byhRS7-SN-38 at nontoxic doses in mice bearing Calu-3 (P≤0.05), Capan-1(P<0.018), BxPC-3 (P<0.005), and COLO 205 tumors (P<0.033) when comparedto nontargeting control ADCs. Mice tolerated a dose of 2×12 mg/kg (SN-38equivalents) with only short-lived elevations in ALT and AST liverenzyme levels. Cynomolgus monkeys infused with 2×0.96 mg/kg exhibitedonly transient decreases in blood counts, although, importantly, thevalues did not fall below normal ranges.

In summary, the anti-Trop-2 hRS7-CL2A-SN-38 ADC provided significant andspecific antitumor effects against a range of human solid tumor types.It was well tolerated in monkeys, with tissue Trop-2 expression similarto humans, at clinically relevant doses.

INTRODUCTION

Successful irinotecan treatment of patients with solid tumors has beenlimited, due in large part to the low conversion rate of the CPT-11prodrug into the active SN-38 metabolite. Others have examinednontargeted forms of SN-38 as a means to bypass the need for thisconversion and to deliver SN-38 passively to tumors. We conjugated SN-38covalently to a humanized anti-Trop-2 antibody, hRS7. This antibody-drugconjugate has specific antitumor effects in a range of s.c. human cancerxenograft models, including non-small cell lung carcinoma, pancreatic,colorectal, and squamous cell lung carcinomas, all at nontoxic doses(e.g., <3.2 mg/kg cumulative SN-38 equivalent dose). Trop-2 is widelyexpressed in many epithelial cancers, but also some normal tissues, andtherefore a dose escalation study in Cynomolgus monkeys was performed toassess the clinical safety of this conjugate. Monkeys tolerated 24 mgSN-38 equivalents/kg with only minor, reversible, toxicities. Given itstumor-targeting and safety profile, hRS7-SN-38 provides a significantimprovement in the management of solid tumors responsive to irinotecan.

Material and Methods

Cell Lines, Antibodies, and Chemotherapeutics—

All human cancer cell lines used in this study were purchased from theAmerican Type Culture Collection. These include Calu-3 (non-small celllung carcinoma), SK-MES-1 (squamous cell lung carcinoma), COLO 205(colonic adenocarcinoma), Capan-1 and BxPC-3 (pancreaticadenocarcinomas), and PC-3 (prostatic adenocarcinomas). Humanized RS7IgG and control humanized anti-CD20 (hA20 IgG, veltuzumab) and anti-CD22(hLL2 IgG, epratuzumab) antibodies were prepared at Immunomedics, Inc.Irinotecan (20 mg/mL) was obtained from Hospira, Inc.

SN-38 ADCs and In Vitro Aspects—

Synthesis of CL2-SN-38 has been described previously (Moon et al., 2008,J Med Chem 51:6916-26). Its conjugation to hRS7 IgG and serum stabilitywere performed as described (Moon et al., 2008, J Med Chem 51:6916-26;Govindan et al., 2009, Clin Chem Res 15:6052-61). Preparations ofCL2A-SN-38 (M.W. 1480) and its hRS7 conjugate, and stability, binding,and cytotoxicity studies, were conducted as described in the precedingExamples.

In Vivo Therapeutic Studies—

For all animal studies, the doses of SN-38 ADCs and irinotecan are shownin SN-38 equivalents. Based on a mean SN-38/IgG substitution ratio of 6,a dose of 500 μg ADC to a 20-g mouse (25 mg/kg) contains 0.4 mg/kg ofSN-38. Irinotecan doses are likewise shown as SN-38 equivalents (i.e.,40 mg irinotecan/kg is equivalent to 24 mg/kg of SN-38).

NCr female athymic nude (nu/nu) mice, 4 to 8 weeks old, and maleSwiss-Webster mice, 10 weeks old, were purchased from Taconic Farms.Tolerability studies were performed in Cynomolgus monkeys (Macacafascicularis; 2.5-4 kg male and female) by SNBL USA, Ltd. Animals wereimplanted subcutaneously with different human cancer cell lines. Tumorvolume (TV) was determined by measurements in 2 dimensions usingcalipers, with volumes defined as: L×w²/2, where L is the longestdimension of the tumor and w is the shortest. Tumors ranged in sizebetween 0.10 and 0.47 cm³ when therapy began. Treatment regimens,dosages, and number of animals in each experiment are described in theResults. The lyophilized hRS7-CL2A-SN-38 and control ADC werereconstituted and diluted as required in sterile saline. All reagentswere administered intraperitoneally (0.1 mL), except irinotecan, whichwas administered intravenously. The dosing regimen was influenced by ourprior investigations, where the ADC was given every 4 days or twiceweekly for varying lengths of time (Moon et al., 2008, J Med Chem51:6916-26; Govindan et al., 2009, Clin Chem Res 15:6052-61). Thisdosing frequency reflected a consideration of the conjugate's serumhalf-life in vitro, to allow a more continuous exposure to the ADC.

Statistics—

Growth curves are shown as percent change in initial TV over time.Statistical analysis of tumor growth was based on area under the curve(AUC). Profiles of individual tumor growth were obtained throughlinear-curve modeling. An f-test was employed to determine equality ofvariance between groups before statistical analysis of growth curves. A2-tailed t-test was used to assess statistical significance between thevarious treatment groups and controls, except for the saline control,where a 1-tailed t-test was used (significance at P<0.05). Statisticalcomparisons of AUC were performed only up to the time that the firstanimal within a group was euthanized due to progression.

Pharmacokinetics and Biodistribution—

¹¹¹In-radiolabeled hRS7-CL2A-SN-38 and hRS7 IgG were injected into nudemice bearing s.c. SK-MES-1 tumors (˜0.3 cm³). One group was injectedintravenously with 20 μCi (250-μg protein) of ¹¹¹In-hRS7-CL2A-SN-38,whereas another group received 20 μCi (250-μg protein) of ¹¹¹In-hRS7IgG. At various timepoints mice (5 per timepoint) were anesthetized,bled via intracardiac puncture, and then euthanized. Tumors and varioustissues were removed, weighed, and counted by γ scintillation todetermine the percentage injected dose per gram tissue (% ID/g). A thirdgroup was injected with 250 μg of unlabeled hRS7-CL2A-SN-38 3 daysbefore the administration of ¹¹¹In-hRS7-CL2A-SN-38 and likewisenecropsied. A 2-tailed t-test was used to compare hRS7-CL2A-SN-38 andhRS7 IgG uptake after determining equality of variance using the f-test.Pharmacokinetic analysis on blood clearance was performed usingWinNonLin software (Parsight Corp.).

Tolerability in Swiss-Webster Mice and Cynomolgus Monkeys—

Briefly, mice were sorted into 4 groups each to receive 2-mL i.p.injections of either a sodium acetate buffer control or 3 differentdoses of hRS7-CL2A-SN-38 (4, 8, or 12 mg/kg of SN-38) on days 0 and 3followed by blood and serum collection, as described in Results.Cynomolgus monkeys (3 male and 3 female; 2.5-4.0 kg) were administered 2different doses of hRS7-CL2A-SN-38. Dosages, times, and number ofmonkeys bled for evaluation of possible hematologic toxicities and serumchemistries are described in the Results.

Results

Stability and Potency of hRS7-CL2A-SN-38—

Two different linkages were used to conjugate SN-38 to hRS7 IgG (FIG.4A). The first is termed CL2-SN-38 and has been described previously(Moon et al., 2008, J Med Chem 51:6916-26; Govindan et al., 2009, ClinChem Res 15:6052-61). A change in the synthesis of CL2 to remove thephenylalanine moiety within the linker was used to produce the CL2Alinker. This change simplified the synthesis, but did not affect theconjugation outcome (e.g., both CL2-SN-38 and CL2A-SN-38 incorporated ˜6SN-38 per IgG molecule). Side-by-side comparisons found no significantdifferences in serum stability, antigen binding, or in vitrocytotoxicity. This result was surprising, since the phenylalanineresidue in CL2 is part of a designed cleavage site for cathepsin B, alysosomal protease.

To confirm that the change in the SN-38 linker from CL2 to CL2A did notimpact in vivo potency, hRS7-CL2A and hRS7-CL2-SN-38 were compared inmice bearing COLO 205 (FIG. 4B) or Capan-1 tumors (FIG. 4C), using 0.4mg or 0.2 mg/kg SN-38 twice weekly×4 weeks, respectively, and withstarting tumors of 0.25 cm³ size in both studies. Both the hRS7-CL2A andCL2-SN-38 conjugates significantly inhibited tumor growth compared tountreated (AUC_(14days) P<0.002 vs. saline in COLO 205 model;AUC_(21days) P<0.001 vs. saline in Capan-1 model), and a nontargetinganti-CD20 control ADC, hA20-CL2A-SN-38 (AUC_(14days) P<0.003 in COLO-205model; AUC_(35days): P<0.002 in Capan-1 model). At the end of the study(day 140) in the Capan-1 model, 50% of the mice treated withhRS7-CL2A-SN-38 and 40% of the hRS7-CL2-SN-38 mice were tumor-free,whereas only 20% of the hA20-ADC-treated animals had no visible sign ofdisease. As demonstrated in FIG. 4, the CL2A linker resulted in asomewhat higher efficacy compared to CL2.

Mechanism of Action—

In vitro cytotoxicity studies demonstrated that hRS7-CL2A-SN-38 had IC₅₀values in the nmol/L range against several different solid tumor lines(Table 4). The IC₅₀ with free SN-38 was lower than the conjugate in allcell lines. Although there was no apparent correlation between Trop-2expression and sensitivity to hRS7-CL2A-SN-38, the IC₅₀ ratio of the ADCversus free SN-38 was lower in the higher Trop-2-expressing cells, mostlikely reflecting the enhanced ability to internalize the drug when moreantigen is present.

SN-38 is known to activate several signaling pathways in cells, leadingto apoptosis (e.g., Cusack et al., 2001, Cancer Res 61:3535-40; Liu etal. 2009, Cancer Lett 274:47-53; Lagadec et al., 2008, Br J Cancer98:335-44). Our initial studies examined the expression of 2 proteinsinvolved in early signaling events (p21^(Waf1/Cip1) and p53) and 1 lateapoptotic event [cleavage of poly-ADP-ribose polymerase (PARP)] in vitro(not shown). In BxPC-3, SN-38 led to a 20-fold increase inp21^(Waf1/Cip1) expression (not shown), whereas hRS7-CL2A-SN-38 resultedin only a 10-fold increase (not shown), a finding consistent with thehigher activity with free SN-38 in this cell line (Table 4). However,hRS7-CL2A-SN-38 increased p21^(Waf1/CiP1) expression in Calu-3 more than2-fold over free SN-38 (not shown).

A greater disparity between hRS7-CL2A-SN-38- and free SN-38-mediatedsignaling events was observed in p53 expression (not shown). In bothBxPC-3 and Calu-3, upregulation of p53 with free SN-38 was not evidentuntil 48 hours, whereas hRS7-CL2A-SN-38 upregulated p53 within 24 hours(not shown). In addition, p53 expression in cells exposed to the ADC washigher in both cell lines compared to SN-38 (not shown). Interestingly,although hRS7 IgG had no appreciable effect on p21^(Waf1/Cip1)expression, it did induce the upregulation of p53 in both BxPC-3 andCalu-3, but only after a 48-hour exposure (not shown). In terms of laterapoptotic events, cleavage of PARP was evident in both cell lines whenincubated with either SN-38 or the conjugate (not shown). The presenceof the cleaved PARP was higher at 24 hours in BxPC-3 (not shown), whichcorrelates with high expression of p21 and its lower IC₅₀. The higherdegree of cleavage with free SN-38 over the ADC was consistent with thecytotoxicity findings.

Efficacy of hRS7-SN-38—

Because Trop-2 is widely expressed in several human carcinomas, studieswere performed in several different human cancer models, which startedusing the hRS7-CL2-SN-38 linkage, but later, conjugates with theCL2A-linkage were used. Calu-3-bearing nude mice given 0.04 mg SN-38/kgof the hRS7-CL2-SN-38 every 4 days×4 had a significantly improvedresponse compared to animals administered the equivalent amount ofnon-targeting hLL2-CL2-SN-38 (TV=0.14±0.22 cm³ vs. 0.80±0.91 cm³,respectively; AUC_(42days) P<0.026; FIG. 5A). A dose-response wasobserved when the dose was increased to 0.4 mg/kg SN-38 (FIG. 5A). Atthis higher dose level, all mice given the specific hRS7 conjugate were“cured” within 28 days, and remained tumor-free until the end of thestudy on day 147, whereas tumors regrew in animals treated with theirrelevant ADC (specific vs. irrelevant AUC_(98days): P=0.05). In micereceiving the mixture of hRS7 IgG and SN-38, tumors progressed >4.5-foldby day 56 (TV=1.10±0.88 cm³; AUC_(56days) P<0.006 vs. hRS7-CL2-SN-38)(FIG. 5A).

Efficacy also was examined in human colonic (COLO 205) and pancreatic(Capan-1) tumor xenografts. In COLO 205 tumor-bearing animals, (FIG.13B), hRS7-CL2-SN-38 (0.4 mg/kg, q4dx8) prevented tumor growth over the28-day treatment period with significantly smaller tumors compared tocontrol anti-CD20 ADC (hA20-CL2-SN-38), or hRS7 IgG (TV=0.16±0.09 cm³,1.19±0.59 cm³, and 1.77±0.93 cm³, respectively; AUC_(28days) P<0.016).

TABLE 4 Expression of Trop-2 in vitro cytotoxicity of SN-38 andhRS7-SN-38 in various solid tumor lines Trop-2 expression via FACSCytotoxicity results Median SN-38 95% CI hRS7-SN-38 95% CI ADC/freefluorescence Percent IC₅₀ IC₅₀ IC₅₀ IC₅₀ SN-38 Cell line (background)positive (nmol/L) (nmol/L) (nmol/L) (nmol/L) ratio Calu-3 282.2 (4.7)99.6% 7.19 5.77-8.95 9.97  8.12-12.25 1.39 COLO 205 141.5 (4.5) 99.5%1.02 0.66-1.57 1.95 1.26-3.01 1.91 Capan-1 100.0 (5.0) 94.2% 3.502.17-5.65 6.99 5.02-9.72 2.00 PC-3 46.2 (5.5) 73.6% 1.86 1.16-2.99 4.242.99-6.01 2.28 SK-MES-1 44.0 (3.5) 91.2% 8.61  6.30-11.76 23.1417.98-29.78 2.69 BxPC-3 26.4 (3.1) 98.3% 1.44 1.04-2.00 4.03 3.25-4.982.80

The MTD of irinotecan (24 mg SN-38/kg, q2dx5) was as effective ashRS7-CL2-SN-38 in COLO 205 cells, because mouse serum can moreefficiently convert irinotecan to SN-38 (Morton et al., 2000, Cancer Res60:4206-10) than human serum, but the SN-38 dose in irinotecan (2,400 μgcumulative) was 37.5-fold greater than with the conjugate (64 μg total).

Animals bearing Capan-1 (FIG. 5C) showed no significant response toirinotecan alone when given at an SN-38-dose equivalent to thehRS7-CL2-SN-38 conjugate (e.g., on day 35, average tumor size was0.04±0.05 cm³ in animals given 0.4 mg SN-38/kg hRS7-SN-38 vs. 1.78±0.62cm³ in irinotecan-treated animals given 0.4 mg/kg SN-38; AUC_(day35)P<0.001; FIG. 5C). When the irinotecan dose was increased 10-fold to 4mg/kg SN-38, the response improved, but still was not as significant asthe conjugate at the 0.4 mg/kg SN-38 dose level (TV=0.17±0.18 cm³ vs.1.69±0.47 cm³, AUC_(day49) P<0.001) (FIG. 5C). An equal dose ofnontargeting hA20-CL2-SN-38 also had a significant antitumor effect ascompared to irinotecan-treated animals, but the specific hRS7 conjugatewas significantly better than the irrelevant ADC (TV=0.17±0.18 cm³ vs.0.80±0.68 cm³, AUC_(day49) P<0.018) (FIG. 5C).

Studies with the hRS7-CL2A-SN-38 ADC were then extended to 2 othermodels of human epithelial cancers. In mice bearing BxPC-3 humanpancreatic tumors (FIG. 13D), hRS7-CL2A-SN-38 again significantlyinhibited tumor growth in comparison to control mice treated with salineor an equivalent amount of nontargeting hA20-CL2A-SN-38 (TV=0.24±0.11cm³ vs. 1.17±0.45 cm³ and 1.05±0.73 cm³, respectively;AUC_(day21)P<0.001), or irinotecan given at a 10-fold higher SN-38equivalent dose (TV=0.27±0.18 cm³ vs. 0.90±0.62 cm³, respectively;AUC_(day25)P<0.004) (FIG. 5D). Interestingly, in mice bearing SK-MES-1human squamous cell lung tumors treated with 0.4 mg/kg of the ADC (FIG.5E), tumor growth inhibition was superior to saline or unconjugated hRS7IgG (TV=0.36±0.25 cm³ vs. 1.02±0.70 cm³ and 1.30±1.08 cm³, respectively;AUC_(28days), P<0.043), but nontargeting hA20-CL2A-SN-38 or the MTD ofirinotecan provided the same antitumor effects as the specifichRS7-SN-38 conjugate (FIG. 5E). In all murine studies, the hRS7-SN-38ADC was well tolerated in terms of body weight loss (not shown).

Biodistribution of hRS7-CL2A-SN-38—

The biodistributions of hRS7-CL2A-SN-38 or unconjugated hRS7 IgG werecompared in mice bearing SK-MES-1 human squamous cell lung carcinomaxenografts (not shown), using the respective ¹¹¹In-labeled substrates. Apharmacokinetic analysis was performed to determine the clearance ofhRS7-CL2A-SN-38 relative to unconjugated hRS7 (not shown). The ADCcleared faster than the equivalent amount of unconjugated hRS7, with theADC exhibiting ˜40% shorter half-life and mean residence time.Nonetheless, this had a minimal impact on tumor uptake (not shown).Although there were significant differences at the 24- and 48-hourtimepoints, by 72 hours (peak uptake) the amounts of both agents in thetumor were similar. Among the normal tissues, hepatic and splenicdifferences were the most striking (not shown). At 24 hourspostinjection, there was >2-fold more hRS7-CL2A-SN-38 in the liver thanhRS7 IgG (not shown). Conversely, in the spleen there was 3-fold moreparental hRS7 IgG present at peak uptake (48-hour timepoint) thanhRS7-CL2A-SN-38 (not shown). Uptake and clearance in the rest of thetissues generally reflected differences in the blood concentration (notshown).

Because twice-weekly doses were given for therapy, tumor uptake in agroup of animals that first received a predose of 0.2 mg/kg (250 μgprotein) of the hRS7 ADC 3 days before the injection of the¹¹¹In-labeled antibody was examined. Tumor uptake of¹¹¹In-hRS7-CL2A-SN-38 in predosed mice was substantially reduced atevery timepoint in comparison to animals that did not receive thepredose (e.g., at 72 hours, predosed tumor uptake was 12.5%±3.8% ID/gvs. 25.4%±8.1% ID/g in animals not given the predose; P=0.0123; notshown). Predosing had no appreciable impact on blood clearance or tissueuptake (not shown). These studies suggest that in some tumor models,tumor accretion of the specific antibody can be reduced by the precedingdose(s), which likely explains why the specificity of a therapeuticresponse could be diminished with increasing ADC doses and why furtherdose escalation is not indicated.

Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster Mice and CynomolgusMonkeys

Swiss-Webster mice tolerated 2 doses over 3 days, each of 4, 8, and 12mg SN-38/kg of the hRS7-CL2A-SN-38, with minimal transient weight loss(not shown). No hematopoietic toxicity occurred and serum chemistriesonly revealed elevated aspartate transaminase (AST, FIG. 6A) and alaninetransaminase (ALT, FIG. 6B). Seven days after treatment, AST rose abovenormal levels (>298 U/L) in all 3 treatment groups (FIG. 6A), with thelargest proportion of mice being in the 2×8 mg/kg group. However, by 15days posttreatment, most animals were within the normal range. ALTlevels were also above the normal range (>77 U/L) within 7 days oftreatment (FIG. 6B) and with evidence of normalization by Day 15. Liversfrom all these mice did not show histologic evidence of tissue damage(not shown). In terms of renal function, only glucose and chloridelevels were somewhat elevated in the treated groups. At 2×8 mg/kg, 5 of7 mice had slightly elevated glucose levels (range of 273-320 mg/dL,upper end of normal 263 mg/dL) that returned to normal by 15 dayspostinjection. Similarly, chloride levels were slightly elevated,ranging from 116 to 127 mmol/L (upper end of normal range 115 mmol/L) inthe 2 highest dosage groups (57% in the 2×8 mg/kg group and 100% of themice in the 2×12 mg/kg group), and remained elevated out to 15 dayspostinjection. This also could be indicative of gastrointestinaltoxicity, because most chloride is obtained through absorption by thegut; however, at termination, there was no histologic evidence of tissuedamage in any organ system examined (not shown).

Because mice do not express Trop-2 identified by hRS7, a more suitablemodel was required to determine the potential of the hRS7 conjugate forclinical use. Immunohistology studies revealed binding in multipletissues in both humans and Cynomolgus monkeys (breast, eye,gastrointestinal tract, kidney, lung, ovary, fallopian tube, pancreas,parathyroid, prostate, salivary gland, skin, thymus, thyroid, tonsil,ureter, urinary bladder, and uterus; not shown). Based on thiscross-reactivity, a tolerability study was performed in monkeys.

The group receiving 2×0.96 mg SN-38/kg of hRS7-CL2A-SN-38 had nosignificant clinical events following the infusion and through thetermination of the study. Weight loss did not exceed 7.3% and returnedto acclimation weights by day 15. Transient decreases were noted in mostof the blood count data (neutrophil and platelet data shown in FIG. 6Cand FIG. 6D), but values did not fall below normal ranges. No abnormalvalues were found in the serum chemistries. Histopathology of theanimals necropsied on day 11 (8 days after last injection) showedmicroscopic changes in hematopoietic organs (thymus, mandibular andmesenteric lymph nodes, spleen, and bone marrow), gastrointestinalorgans (stomach, duodenum, jejunum, ileum, cecum, colon, and rectum),female reproductive organs (ovary, uterus, and vagina), and at theinjection site. These changes ranged from minimal to moderate and werefully reversed at the end of the recovery period (day 32) in alltissues, except in the thymus and gastrointestinal tract, which weretrending towards full recovery at this later timepoint (not shown).

At the 2×1.92 mg SN-38/kg dose level of the conjugate, there was 1 deatharising from gastrointestinal complications and bone marrow suppression,and other animals within this group showed similar, but more severeadverse events than the 2×0.96 mg/kg group (not shown). These dataindicate that dose-limiting toxicities were identical to that ofirinotecan; namely, intestinal and hematologic. Thus, the MTD forhRS7-CL2A-SN-38 lies between 2×0.96 and 1.92 mg SN-38/kg, whichrepresents a human equivalent dose of 2×0.3 to 0.6 mg/kg SN-38.

DISCUSSION

Trop-2 is a protein expressed on many epithelial tumors, including lung,breast, colorectal, pancreas, prostate, and ovarian cancers, making it apotentially important target for delivering cytotoxic agents (Ohmachi etal., 2006, Clin Cancer Res 12:3057-63; Fong et al., 2008, Br J Cancer99:1290-95; Cubas et al., 2009, Biochim Biophys Acta 1796:309-14). TheRS7 antibody internalizes when bound to Trop-2 (Shih et al., 1995,Cancer Res 55:5857s-63s), which enables direct intracellular delivery ofcytotoxics.

SN-38 is a potent topoisomerase-I inhibitor, with IC₅₀ values in thenanomolar range in several cell lines. It is the active form of theprodrug, irinotecan, that is used for the treatment of colorectalcancer, and which also has activity in lung, breast, and brain cancers.We reasoned that a directly targeted SN-38, in the form of an ADC, wouldbe a significantly improved therapeutic over CPT-11, by overcoming thelatter's low and patient-variable bioconversion to active SN-38(Mathijssen et al., 2001, Clin Cancer Res 7:2182-94).

The Phe-Lys peptide inserted in the original CL2 derivative allowed forpossible cleavage via cathepsin B. To simplify the synthetic process, inCL2A the phenylalanine was eliminated, and thus the cathepsin B cleavagesite was removed. Interestingly, this product had a better-definedchromatographic profile compared to the broad profile obtained with CL2(not shown), but more importantly, this change had no impact on theconjugate's binding or stability, and surprisingly produced a smallincrease in potency in side-by-side testing.

In vitro cytotoxicity of hRS7 ADC against a range of solid tumor celllines consistently had IC₅₀ values in the nmol/L range. However, cellsexposed to free SN-38 demonstrated a lower IC₅₀ value compared to theADC. This disparity between free and conjugated SN-38 was also reportedfor ENZ-2208 (Sapra et al., 2008, Clin Cancer Res 14:1888-96, Zhao etal., 2008, Bioconjug Chem 19:849-59) and NK012 (Koizumi et al., 2006,Cancer Res 66:10048-56). ENZ-2208 utilizes a branched PEG to link about3.5 to 4 molecules of SN-38 per PEG, whereas NK012 is a micellenanoparticle containing 20% SN-38 by weight. With our ADC, thisdisparity (i.e., ratio of potency with free vs. conjugated SN-38)decreased as the Trop-2 expression levels increased in the tumor cells,suggesting an advantage to targeted delivery of the drug. In terms ofViir0 serum stability, both the CL2- and CL2A-SN-38 forms of hRS7-SN-38yielded a t/½ of −20 hours, which is in contrast to the short t/½ of12.3 minutes reported for ENZ-2208 (Zhao et al., 2008, Bioconjug Chem19:849-59), but similar to the 57% release of SN-38 from NK012 underphysiological conditions after 24 hours (Koizumi et al., 2006, CancerRes 66:10048-56). Treatment of tumor-bearing mice with hRS7-SN-38(either with CL2-SN-38 or CL2A-SN-38) significantly inhibited tumorgrowth in 5 different tumor models. In 4 of them, tumor regressions wereobserved, and in the case of Calu-3, all mice receiving the highest doseof hRS7-SN-38 were tumor-free at the conclusion of study. Unlike inhumans, irinotecan is very efficiently converted to SN-38 by a plasmaesterase in mice, with a greater than 50% conversion rate, and yieldinghigher efficacy in mice than in humans (Morton et al., 2000, Cancer Res60:4206-10; Furman et al., 1999, J Clin Oncol 17:1815-24). Whenirinotecan was administered at 10-fold higher or equivalent SN-38levels, hRS7-SN-38 was significantly better in controlling tumor growth.Only when irinotecan was administered at its MTD of 24 mg/kg q2dx5(37.5-fold more SN-38) did it equal the effectiveness of hRS7-SN-38. Inpatients, we would expect this advantage to favor hRS7-CL2A-SN-38 evenmore, because the bioconversion of irinotecan would be substantiallylower.

We also showed in some antigen-expressing cell lines, such as SK-MES-1,that using an antigen-binding ADC does not guarantee better therapeuticresponses than a nonbinding, irrelevant conjugate. This is not anunusual or unexpected finding. Indeed, the nonbinding SN-38 conjugatesmentioned earlier enhance therapeutic activity when compared toirinotecan, and so an irrelevant IgG-SN-38 conjugate is expected to havesome activity. This is related to the fact that tumors have immature,leaky vessels that allow the passage of macromolecules better thannormal tissues (Jain, 1994, Sci Am 271:58-61). With our conjugate, 50%of the SN-38 will be released in ˜13 hours when the pH is lowered to alevel mimicking lysosomal levels (e.g., pH 5.3 at 37° C.; data notshown), whereas at the neutral pH of serum, the release rate is reducednearly 2-fold. If an irrelevant conjugate enters an acidic tumormicroenvironment, it is expected to release some SN-38 locally. Otherfactors, such as tumor physiology and innate sensitivities to the drug,will also play a role in defining this “baseline” activity. However, aspecific conjugate with a longer residence time should have enhancedpotency over this baseline response as long as there is ample antigen tocapture the specific antibody. Biodistribution studies in the SK-MES-1model also showed that if tumor antigen becomes saturated as aconsequence of successive dosing, tumor uptake of the specific conjugateis reduced, which yields therapeutic results similar to that found withan irrelevant conjugate.

Although it is challenging to make direct comparisons between our ADCand the published reports of other SN-38 delivery agents, some generalobservations can be made. In our therapy studies, the highest individualdose was 0.4 mg/kg of SN-38. In the Calu-3 model, only 4 injections weregiven for a total cumulative dose of 1.6 mg/kg SN-38 or 32 μg SN-38 in a20 g mouse. Multiple studies with ENZ-2208 were done using its MTD of 10mg/kg×5 (Sapra et al., 2008, Clin Cancer Res 14:1888-96; Pastorini etal., 2010, Clin Cancer Res 16:4809-21), and preclinical studies withNK012 involved its MTD of 30 mg/kg×3 (Koizumi et al., 2006, Cancer Res66:10048-56). Thus, significant antitumor effects were obtained withhRS7-SN-38 at 30-fold and 55-fold less SN-38 equivalents than thereported doses in ENZ-2208 and NK012, respectively. Even with 10-foldless hRS7 ADC (0.04 mg/kg), significant antitumor effects were observed,whereas lower doses of ENZ-2208 were not presented, and when the NK012dose was lowered 4-fold to 7.5 mg/kg, efficacy was lost (Koizumi et al.,2006, Cancer Res 66:10048-56). Normal mice showed no acute toxicity witha cumulative dose over 1 week of 24 mg/kg SN-38 (1,500 mg/kg of theconjugate), indicating that the MTD was higher. Thus, tumor-bearinganimals were effectively treated with 7.5- to 15-fold lower amounts ofSN-38 equivalents.

Biodistribution studies revealed the hRS7-CL2A-SN-38 had similar tumoruptake as the parental hRS7 IgG, but cleared substantially faster with2-fold higher hepatic uptake, which may be due to the hydrophobicity ofSN-38. With the ADC being cleared through the liver, hepatic andgastrointestinal toxicities were expected to be dose limiting. Althoughmice had evidence of increased hepatic transaminases, gastrointestinaltoxicity was mild at best, with only transient loss in weight and noabnormalities noted upon histopathologic examination. Interestingly, nohematological toxicity was noted. However, monkeys showed an identicaltoxicity profile as expected for irinotecan, with gastrointestinal andhematological toxicity being dose-limiting.

Because Trop-2 recognized by hRS7 is not expressed in mice, it wasimportant to perform toxicity studies in monkeys that have a similartissue expression of Trop-2 as humans. Monkeys tolerated 0.96 mg/kg/dose(˜12 mg/m²) with mild and reversible toxicity, which extrapolates to ahuman dose of ˜0.3 mg/kg/dose (˜11 mg/m²). In a Phase I clinical trialof NK012, patients with solid tumors tolerated 28 mg/m² of SN-38 every 3weeks with Grade 4 neutropenia as dose-limiting toxicity (DLT; Hamaguchiet al., 2010, Clin Cancer Res 16:5058-66). Similarly, Phase I clinicaltrials with ENZ-2208 revealed dose-limiting febrile neutropenia, with arecommendation to administer 10 mg/m² every 3 weeks or 16 mg/m² ifpatients were administered G-CSF (Kurzrock et al., AACR-NCI-EORTCInternational Conference on Molecular Targets and Cancer Therapeutics;2009 Nov. 15-19: Boston, Mass.; Poster No C216: Patnaik et al.,AACR-NCI-EORTC International Conference on Molecular Targets and CancerTherapeutics; 2009 Nov. 15-19; Boston, Mass.; Poster No C221). Becausemonkeys tolerated a cumulative human equivalent dose of 22 mg/m², itappears that even though hRS7 binds to a number of normal tissues, theMTD for a single treatment of the hRS7 ADC could be similar to that ofthe other nontargeting SN-38 agents. Indeed, the specificity of theanti-Trop-2 antibody did not appear to play a role in defining the DLT,because the toxicity profile was similar to that of irinotecan. Moreimportantly, if antitumor activity can be achieved in humans as in micethat responded with human equivalent dose of just at 0.03 mg SN-38equivalents/kg/dose, then significant antitumor responses may berealized clinically.

In conclusion, toxicology studies in monkeys, combined with in vivohuman cancer xenograft models in mice, have indicated that this ADCtargeting Trop-2 is an effective therapeutic in several tumors ofdifferent epithelial origin.

Example 4. Cell Binding Assay of Anti-Trop-2 Antibodies

Two different murine monoclonal antibodies against human Trop-2 wereobtained for ADC conjugation. The first, 162-46.2, was purified from ahybridoma (ATCC, HB-187) grown up in roller-bottles. A second antibody,MAB650, was purchased from R&D Systems (Minneapolis, Minn.). For acomparison of binding, the Trop-2 positive human gastric carcinoma,NCI-N87, was used as the target. Cells (1.5×10⁵/well) were plated into96-well plates the day before the binding assay. The following morning,a dose/response curve was generated with 162-46.2, MAB650, and murineRS7 (0.03 to 66 nM). These primary antibodies were incubated with thecells for 1.5 h at 4° C. Wells were washed and an anti-mouse-HRPsecondary antibody was added to all the wells for 1 h at 4° C. Wells arewashed again followed by the addition of a luminescence substrate.Plates were read using Envision plate reader and values are reported asrelative luminescent units.

All three antibodies had similar K_(D)-values of 0.57 nM for RS7, 0.52nM for 162-46.2 and 0.49 nM for MAB650. However, when comparing themaximum binding (B_(max)) of 162-46.2 and MAB650 to RS7 they werereduced by 25% and 50%, respectively (B_(Max) 11,250 for RS7, 8,471 for162-46.2 and 6,018 for MAB650) indicating different binding propertiesin comparison to RS7.

Example 5. Cytotoxicity of Anti-Trop-2 ADC (MAB650-SN-38)

A novel anti-Trop-2 ADC was made with SN-38 and MAB650, yielding a meandrug to antibody substitution ratio of 6.89. Cytotoxicity assays wereperformed to compare the MAB650-SN-38 and hRS7-SN-38 ADCs using twodifferent human pancreatic adenocarcinoma cell lines (BxPC-3 andCapan-1) and a human triple negative breast carcinoma cell line(MDA-MB-468) as targets.

One day prior to adding the ADCs, cells were harvested from tissueculture and plated into 96-well plates. The next day cells were exposedto hRS7-SN-38, MAB650-SN-38, and free SN-38 at a drug range of3.84×10⁻¹² to 2.5×10⁻⁷ M. Unconjugated MAB650 was used as a control atprotein equivalent doses as the MAB650-SN-38. Plates were incubated at37° C. for 96 h. After this incubation period, an MTS substrate wasadded to all of the plates and read for color development at half-hourintervals until an OD_(492nm) of approximately 1.0 was reached for theuntreated cells. Growth inhibition was measured as a percent of growthrelative to untreated cells using Microsoft Excel and Prism software(non-linear regression to generate sigmoidal dose response curves whichyield IC₅₀-values.

As shown in FIG. 7, hRS7-SN-38 and MAB650-SN-38 had similargrowth-inhibitory effects with IC₅₀-values in the low nM range which istypical for SN-38-ADCs in these cell lines. In the human Capan-1pancreatic adenocarcinoma cell line (FIG. 7A), the hRS7-SN-38 ADC showedan IC₅₀ of 3.5 nM, compared to 4.1 nM for the MAB650-SN-38 ADC and 1.0nM for free SN-38. In the human BxPC-3 pancreatic adenocarcinoma cellline (FIG. 7B), the hRS7-SN-38 ADC showed an IC₅₀ of 2.6 nM, compared to3.0 nM for the MAB650-SN-38 ADC and 1.0 nM for free SN-38. In the humanNCI-N87 gastric adenocarcinoma cell line (FIG. 7C), the hRS7-SN-38 ADCshowed an IC₅₀ of 3.6 nM, compared to 4.1 nM for the MAB650-SN-38 ADCand 4.3 nM for free SN-38.

In summary, in these in vitro assays, the SN-38 conjugates of twoanti-Trop-2 antibodies, hRS7 and MAB650, showed equal efficacies againstseveral tumor cell lines, which was similar to that of free SN-38.Because the targeting function of the anti-Trop-2 antibodies would be amuch more significant factor in vivo than in vitro, the data supportthat anti-Trop-2-SN-38 ADCs as a class would be highly efficacious invivo, as demonstrated in the Examples above for hRS7-SN-38.

Example 6. Cytotoxicity of Anti-Trop-2 ADC (162-46.2-SN-38)

A novel anti-Trop-2 ADC was made with SN-38 and 162-46.2, yielding adrug to antibody substitution ratio of 6.14. Cytotoxicity assays wereperformed to compare the 162-46.2-SN-38 and hRS7-SN-38 ADCs using twodifferent Trop-2-positive cell lines as targets, the BxPC-3 humanpancreatic adenocarcinoma and the MDA-MB-468 human triple negativebreast carcinoma.

One day prior to adding the ADC, cells were harvested from tissueculture and plated into 96-well plates at 2000 cells per well. The nextday cells were exposed to hRS7-SN-38, 162-46.2-SN-38, or free SN-38 at adrug range of 3.84×10⁻¹² to 2.5×10⁻⁷M. Unconjugated 162-46.2 and hRS7were used as controls at the same protein equivalent doses as the162-46.2-SN-38 and hRS7-SN-38, respectively. Plates were incubated at37° C. for 96 h. After this incubation period, an MTS substrate wasadded to all of the plates and read for color development at half-hourintervals until untreated control wells had an OD_(492nm) reading ofapproximately 1.0. Growth inhibition was measured as a percent of growthrelative to untreated cells using Microsoft Excel and Prism software(non-linear regression to generate sigmoidal dose response curves whichyield IC₅₀-values).

As shown in FIG. 8A and FIG. 8B, the 162-46.2-SN-38 ADC had a similarIC₅₀-values when compared to hRS7-SN-38. When tested against the BxPC-3human pancreatic adenocarcinoma cell line (FIG. 8A), hRS7-SN-38 had anIC₅₀ of 5.8 nM, compared to 10.6 nM for 162-46.2-SN-38 and 1.6 nM forfree SN-38. When tested against the MDA-MB-468 human breastadenocarcinoma cell line (FIG. 8B), hRS7-SN-38 had an IC₅₀ of 3.9 nM,compared to 6.1 nM for 162-46.2-SN-38 and 0.8 nM for free SN-38. Thefree antibodies alone showed little cytotoxicity to either Trop-2positive cancer cell line.

In summary, comparing the efficacies in vitro of three differentanti-Trop-2 antibodies conjugated to the same cytotoxic drug, all threeADCs exhibited equivalent cytotoxic effects against a variety of Trop-2positive cancer cell lines. These data support that the class ofanti-Trop-2 antibodies, incorporated into drug-conjugated ADCs, areeffective anti-cancer therapeutic agents for Trop-2 expressing solidtumors.

Example 7. Clinical Trials With IMMU-132 Anti-Trop-2 ADC Comprising hRS7Antibody Conjugated to SN-38 SUMMARY

The present Example reports results from a phase I clinical trial andongoing phase II extension with IMMU-132, an ADC of the internalizing,humanized, hRS7 anti-Trop-2 antibody conjugated by a pH-sensitive linkerto SN-38 (mean drug-antibody ratio=7.6). Trop-2 is a type Itransmembrane, calcium-transducing, protein expressed at high density(−1×10⁵), frequency, and specificity by many human carcinomas, withlimited normal tissue expression. Preclinical studies in nude micebearing Capan-1 human pancreatic tumor xenografts have revealed IMMU-132is capable of delivering as much as 120-fold more SN-38 to tumor thanderived from a maximally tolerated irinotecan therapy.

The present Example reports the initial Phase I trial of 25 patients whohad failed multiple prior therapies (some including topoisomerase-I/IIinhibiting drugs), and the ongoing Phase II extension now reporting on69 patients, including in colorectal (CRC), small-cell and non-smallcell lung (SCLC, NSCLC, respectively), triple-negative breast (TNBC),pancreatic (PDC), esophageal, and other cancers.

As discussed in detail below, Trop-2 was not detected in serum, but wasstrongly expressed (>2⁺ immunohistochemical staining) in most archivedtumors. In a 3+3 trial design, IMMU-132 was given on days 1 and 8 inrepeated 21-day cycles, starting at 8 mg/kg/dose, then 12 and 18 mg/kgbefore dose-limiting neutropenia. To optimize cumulative treatment withminimal delays, phase II is focusing on 8 and 10 mg/kg (n=30 and 14,respectively). In 49 patients reporting related AE at this time,neutropenia >Grade 3 occurred in 28% (4% Grade 4). Most commonnon-hematological toxicities initially in these patients have beenfatigue (55%; ≥G3=9%), nausea (53%; ≥G3=0%), diarrhea (47%; ≥G3=9%),alopecia (40%), and vomiting (32%; ≥G3=2%); alopecia also occurredfrequently. Homozygous UGT1A1*28/*28 was found in 6 patients, 2 of whomhad more severe hematological and GI toxicities.

In the Phase I and the expansion phases, there are now 48 patients(excluding PDC) who are assessable by RECIST/CT for best response. Seven(15%) of the patients had a partial response (PR), including patientswith CRC (N=1), TNBC (N=2), SCLC (N=2), NSCLC (N=1), and esophagealcancers (N=1), and another 27 patients (56%) had stable disease (SD),for a total of 38 patients (79%) with disease response; 8 of 13CT-assessable PDC patients (62%) had SD, with a median time toprogression (TTP) of 12.7 wks compared to 8.0 weeks in their last priortherapy. The TTP for the remaining 48 patients is 12.6+ wks (range 6.0to 51.4 wks). Plasma CEA and CA19-9 correlated with responses who hadelevated titers of these antigens in their blood. No anti-hRS7 oranti-SN-38 antibodies were detected despite dosing over months.

The conjugate cleared from the serum within 3 days, consistent with invivo animal studies where 50% of the SN-38 was released daily, with >95%of the SN-38 in the serum being bound to the IgG in a non-glucoronidatedform, and at concentrations as much as 100-fold higher than SN-38reported in patients given irinotecan. These results show that thehRS7-SN-38-containing ADC is therapeutically active in metastatic solidcancers, with manageable diarrhea and neutropenia.

Pharmacokinetics

Two ELISA methods were used to measure the clearance of the IgG (capturewith anti-hRS7 idiotype antibody) and the intact conjugate (capture withanti-SN-38 IgG/probe with anti-hRS7 idiotype antibody). SN-38 wasmeasured by HPLC. Total IMMU-132 fraction (intact conjugate) clearedmore quickly than the IgG (not shown), reflecting known gradual releaseof SN-38 from the conjugate. HPLC determination of SN-38 (Unbound andTOTAL) showed >95% the SN-38 in the serum was bound to the IgG. Lowconcentrations of SN-38G suggest SN-38 bound to the IgG is protectedfrom glucoronidation. Comparison of ELISA for conjugate and SN-38 HPLCrevealed both overlap, suggesting the ELISA is a surrogate formonitoring SN-38 clearance.

A summary of the dosing regiment and patient pool is provided in Table5.

TABLE 5 Clinical Trial Parameters Dosing regimen Once weekly for 2 weeksadministered every 21 days for up to 8 cycles. In the initialenrollment, the planned dose was delayed and reduced if ≥Grade 2treatment-related toxicity; protocol was amended to dose delay andreduction only in the event of ≥Grade 3 toxicity. Dose level cohorts 8,12, 18 mg/kg; later reduced to an intermediate dose level of 10 mg/kg.Cohort size Standard Phase I [3 + 3] design; expansion includes ~15patients in select cancers. DLT Grade 4 ANC ≥7 d; ≥Grade 3 febrileneutropenia of any duration; G4 Plt ≥5 d; G4 Hgb; Grade 4 N/V/D anyduration/G3 N/V/D for >48 h; G3 infusion-related reactions; related ≥G3non-hematological toxicity. Maximum Maximum dose where ≥2/6 patientstolerate 1^(st) 21-d cycle w/o delay or Acceptable Dose reduction or ≥G3toxicity. (MAD) Patients Metastatic colorectal, pancreas, gastric,esophageal, lung (NSCLC, SCLC), triple-negative breast (TNBC), prostate,ovarian, renal, urinary bladder, head/neck, hepatocellular.Refractory/relapsed after standard treatment regimens for metastaticcancer. Prior irinotecan-containing therapy NOT required for enrollment.No bulky lesion >5 cm. Must be 4 weeks beyond any major surgery, and 2weeks beyond radiation or chemotherapy regimen. Gilbert's disease orknown CNS metastatic disease are excluded.

Clinical Trial Status

A total of 69 patients (including 25 patients in Phase I) with diversemetastatic cancers having a median of 3 prior therapies were reported.Eight patients had clinical progression and withdrew before CTassessment. Thirteen CT-assessable pancreatic cancer patients wereseparately reported. The median TTP (time to progression) in PDCpatients was 11.9 wks (range 2 to 21.4 wks) compared to median 8 wks TTPfor the preceding last therapy.

A total of 48 patients with diverse cancers had at least 1 CT-assessmentfrom which Best Response (FIG. 9) and Time to Progression (TTP; FIG. 20)were determined. To summarize the Best Response data, of 8 assessablepatients with TNBC (triple-negative breast cancer), there were 2 PR(partial response), 4 SD (stable disease) and 2 PD (progressive disease)for a total response [PR+SD] of 6/8 (75%). For SCLC (small cell lungcancer), of 4 assessable patients there were 2 PR, 0 SD and 2 PD for atotal response of 2/4 (50%). For CRC (colorectal cancer), of 18assessable patients there were 1 PR, 11 SD and 6 PD for a total responseof 12/18 (67%). For esophageal cancer, of 4 assessable patients therewere 1 PR, 2 SD and 1 PD for a total response of 3/4 (75%). For NSCLC(non-small cell lung cancer), of 5 assessable patients there were 1 PR,3 SD and 1 PD for a total response of 4/5 (80%). Over all patientstreated, of 48 assessable patients there were 7 PR, 27 SD and 14 PD fora total response of 34/48 (71%). These results demonstrate that theanti-Trop-2 ADC (hRS7-SN-38) showed significant clinical efficacyagainst a wide range of solid tumors in human patients.

The reported side effects of therapy (adverse events) are summarized inTable 6. As apparent from the data of Table 6, the therapeutic efficacyof hRS7-SN-38 was achieved at dosages of ADC showing an acceptably lowlevel of adverse side effects.

TABLE 6 Related Adverse Events Listing for IMMU-132-01 Criteria: Total≥10% or ≥Grade 3 N = 47 patients TOTAL Grade 3 Grade 4 Fatigue 55% 4(9%) 0 Nausea 53% 0 0 Diarrhea 47% 4 (9%) 0 Neutropenia 43% 11 (24%) 2(4%) Alopecia 40% — — Vomiting 32% 1 (2%) 0 Anemia 13% 2 (4%) 0Dysgeusia 15% 0 0 Pyrexia 13% 0 0 Abdominal pain 11% 0 0 Hypokalemia 11%1 (2%) 0 WBC Decrease  6% 1 (2%) 0 Febrile Neutropenia  6% 1 (2%) 2 (4%)Deep vein thrombosis  2% 1 (2%) 0 Grading by CTCAE v 4.0

The study reported in Table 6 has continued, with 261 patients enrolledto date. The results (not shown) have generally followed along the linesindicated in Table 6, with only neutropenia showing an incidence ofGrade 3 or higher adverse events of over 10% of the patients tested. Forall other adverse events, the incidence of Grade 3 or higher responseswas less than 10%. This distinguishes the instant ADCs from the greatmajority of ADCs and in certain embodiments, the claimed methods andcompositions relate to anti-Trop-2 ADCs that show efficacy in diversesolid tumors, with an incidence of Grade 3 or higher adverse events ofless than 10% of patients for all adverse events other than neutropenia.In a follow-up study, in a total of 421 samples from 121 patients withbaseline and at least one follow-up sample available, no anti-hRS7 oranti-SN-38 antibody response has been detected, despite repeated cyclesof treatment.

Exemplary partial responses to the anti-Trop-2 ADC were confirmed by CTdata (not shown). As an exemplary PR in CRC, a 62 year-old woman firstdiagnosed with CRC underwent a primary hemicolectomy. Four months later,she had a hepatic resection for liver metastases and received 7 mos oftreatment with FOLFOX and 1 mo SFU. She presented with multiple lesionsprimarily in the liver (3+ Trop-2 by immunohistology), entering thehRS7-SN-38 trial at a starting dose of 8 mg/kg about 1 year afterinitial diagnosis. On her first CT assessment, a PR was achieved, with a37% reduction in target lesions (not shown). The patient continuedtreatment, achieving a maximum reduction of 65% decrease after 10 monthsof treatment (not shown) with decrease in CEA from 781 ng/mL to 26.5ng/mL), before progressing 3 months later.

As an exemplary PR in NSCLC, a 65 year-old male was diagnosed with stageIIIB NSCLC (sq. cell). Initial treatment of caboplatin/etoposide (3 mo)in concert with 7000 cGy XRT resulted in a response lasting 10 mo. Hewas then started on Tarceva maintenance therapy, which he continueduntil he was considered for IMMU-132 trial, in addition to undergoing alumbar laminectomy. He received first dose of IMMU-132 after 5 months ofTarceva, presenting at the time with a 5.6 cm lesion in the right lungwith abundant pleural effusion. He had just completed his 6^(th) dosetwo months later when the first CT showed the primary target lesionreduced to 3.2 cm (not shown).

As an exemplary PR in SCLC, a 65 year-old woman was diagnosed withpoorly differentiated SCLC. After receiving carboplatin/etoposide(Topoisomerase-II inhibitor) that ended after 2 months with no response,followed with topotecan (Topoisomerase-I inhibitor) that ended after 2months, also with no response, she received local XRT (3000 cGy) thatended 1 month later. However, by the following month progression hadcontinued. The patient started with IMMU-132 the next month (12 mg/kg;reduced to 6.8 mg/kg; Trop-2 expression 3+), and after two months ofIMMU-132, a 38% reduction in target lesions, including a substantialreduction in the main lung lesion occurred (not shown). The patientprogressed 3 months later after receiving 12 doses.

These results are significant in that they demonstrate that theanti-Trop-2 ADC was efficacious, even in patients who had failed orprogressed after multiple previous therapies. In conclusion, at thedosages used, the primary toxicity was a manageable neutropenia, withfew Grade 3 toxicities. IMMU-132 showed evidence of activity (PR anddurable SD) in relapsed/refractory patients with triple-negative breastcancer, small cell lung cancer, non-small cell lung cancer, colorectalcancer and esophageal cancer, including patients with a previous historyof relapsing on topoisomerase-I inhibitor therapy. These results showefficacy of the anti-Trop-2 ADC in a wide range of cancers that areresistant to existing therapies.

Example 8. Comparative Efficacy of Different Anti-Trop-2 ADCs

The therapeutic efficacy of a murine anti-Trop-2 monoclonal antibody(162-46.2) conjugated with SN-38 was compared to hRS7-SN-38antibody-drug conjugate (ADC) in mice bearing human gastric carcinomaxenografts (NCI-N87). NCI-N87 cells were expanded in tissue culture andharvested with trypsin/EDTA. Female athymic nude mice were injected s.c.with 200 □L of NCI-N87 cell suspension mixed 1:1 with matrigel such that1×10⁷ cells was administered to each mouse. Once tumors reachedapproximately 0.25 cm³ in size (6 days later), the animals were dividedup into seven different treatment groups of nine mice each. For theSN-38 ADCs, mice received 500 μg i.v. injections once a week for twoweeks. Control mice received the non-tumor targeting hA20-SN-38 ADC atthe same dose/schedule. A final group of mice received only saline andserved as the untreated control. Tumors were measured and mice weighedtwice a week. Mice were euthanized for disease progression if theirtumor volumes exceeded 1.0 cm³ in size.

Mean tumor volumes for the SN-38-ADC treated mice are shown in FIG. 11.As determined by area under the curve (AUC), both hRS7-SN-38 and162-46.2-SN-38 significantly inhibited tumor growth when compared tosaline and hA20-SN-38 control mice (P<0.001). Treatment with hRS7-SN-38achieved stable disease in 7 of 9 mice with mean time to tumorprogression (TTP) of 18.4±3.3 days. Mice treated with 162-46.2-SN-38achieved a positive response in 6 of 9 mice with the remaining 3achieving stable disease. Mean TTP was 24.2±6.0 days, which issignificantly longer than hRS7-SN-38 treated animals (P=0.0382). Theseresults confirm the in vivo efficacy of different anti-Trop-2 ADCs fortreatment of human gastric carcinoma.

Example 9. Treatment of Patients with Advanced, Metastatic PancreaticCancer With Anti-Trop-2 ADC SUMMARY

IMMU-132 (hRS7-SN-38) is an anti-Trop-2 ADC comprising the cancer cellinternalizing, humanized, anti-Trop-2 hRS7 antibody, conjugated by apH-sensitive linker to SN-38, the active metabolite of irinotecan, at amean drug-antibody ratio of 7.6. Trop-2 is a type-I transmembrane,calcium-transducing protein expressed at high density, frequency, andspecificity in many epithelial cancers, including pancreatic ductaladenocarcinoma, with limited normal tissue expression. All 29 pancreatictumor microarray specimens tested were Trop-2-positive byimmunohistochemistry, and human pancreatic cancer cell lines were foundto express 115k-891k Trop-2 copies on the cell membrane.

We reported above the results from the IMMU-132 Phase I study enrollingpatients with 13 different tumor types using a 3+3 design. The Phase Idose-limiting toxicity was neutropenia. Over 80% of 24 assessablepatients in this study had long-term stable disease, with partialresponses (RECIST) observed in patients with colorectal (CRC),triple-negative breast (TNBC), small-cell and non-small cell lung (SCLC,NSCLC), and esophageal (EAC) cancers. The present Example reports theresults from the IMMU-132 Phase I/II study cohort of patients withmetastatic PDC. Patients with PDC who failed a median of 2 priortherapies (range 1-5) were given IMMU-132 on days 1 and 8 in repeated21-day cycles.

In the subgroup of PDC patients (N=15), 14 received priorgemcitabine-containing regimens. Initial toxicity data from 9 patientsfound neutropenia [3 of 9≥G3, 33%; and 1 case of G4 febrileneutropenia), which resulted in dose delays or dose reductions. Twopatients had Grade 3 diarrhea; no patient had Grade 3-4 nausea orvomiting. Alopecia (Grades 1-2) occurred in 5 of 9 patients. Bestresponse was assessable in 13 of 14 patients, with 8 stable disease for8 to 21.4 wks (median 12.7 wks; 11.9 wks all 14 patients). One patientwho is continuing treatment has not yet had their first CT assessment.Five had progressive disease by RECIST; 1 withdrew after just 1 dose dueto clinical progression and was not assessable. Serum CA19-9 titersdecreased in 3 of the patients with stable disease by 23 to 72%. Despitemultiple administrations, none of the patients developed an antibodyresponse to IMMU-132 or SN-38. Peak and trough serum samples showed thatIMMU-132 cleared more quickly than the IgG, which is expected based onthe known local release of SN-38 within the tumor cell. Concentrationsof SN-38-bound to IgG in peak samples from one patient given 12 mg/kg ofIMMU-132 showed levels of 4000 ng/mL, which is 40-times higher than theSN-38 titers reported in patients given irinotecan therapy.

We conclude that IMMU-132 is active (long-term stable disease) in 62%(8/13) of PDC patients who failed multiple prior therapies, withmanageable neutropenia and little GI toxicity. Advanced PDC patients canbe given repeated treatment cycles (>6) of 8-10 mg/kg IMMU-132 on days 1and 8 of a 21-day cycle, with some dose adjustments or growth factorsupport for neutropenia in subsequent treatment cycles. These resultsagree with the findings in patients with advanced CRC, TNBC, SCLC,NSCLC, EAC who have shown partial responses and long-term stable diseasewith IMMU-132 administration. In summary, monotherapy IMMU-132 is anovel, efficacious treatment regimen for patients with PDC, includingthose with tumors that were previously resistant to other therapeuticregimens for PDC.

Methods and Results

Trop-2 Expression—

The expression of Trop-2 on the surface of various cancer cell lines wasdetermined by flow cytometry using QUANTBRITE® PE beads. The results fornumber of Trop-2 molecules detected in the different cell lines was:BxPC-3 pancreatic cancer (891,000); NCI-N87 gastric cancer (383,000);MDA-MB-468 breast cacner (341,000); SK-IVIES-1 squamous cell lung cancer(27,000); Capan-1 pancreatic cancer (115,000); AGS gastric cancer(78,000) COLO 205 colon cancer (52,000). Trop-2 expression was alsoobserved in 29 of 29 (100%) tissue microarrays of pancreaticadenocarcinoma (not shown).

Sn-38 Accumulation—

SN-38 accumulation was determined in nude mice bearing Capan-1 humanpancreatic cancer xenografts (−0.06-0.27 g). Mice were injected IV withirinotecan 40 mg/kg (773 μg; Total SN-38 equivalents=448 μg). This doseis MTD in mice. Human dose equivalent=3.25 mg/kg or −126 mg/m². Or micewere injected IV with IMMU-132 1.0 mg (SN-38:antibody ratio=7.6; SN-38equivalents=20 μg). This dose is well below the MTD in mice. Humanequivalent dose ˜4 mg/kg IMMU-132 (−80 μg/kg SN-38 equivalents).Necropsies were performed on 3 animals per interval, in irinotecaninjected mice at 5 min, 1, 2, 6 and 24 hours or in IMMU-132 injectedmice at 1, 6, 24, 48 and 72 h. Tissues were extracted and analyzed byreversed-phase HPLC analysis for SN-38, SN-38G, and irinotecan. Extractsfrom IMMU-132-treated animals also were acid hydrolyzed to release SN-38from the conjugate (i.e., SN-38 (TOTAL]). The results, shown in FIG. 12,demonstrate that the IMMU-132 ADC has the potential to deliver 120 timesmore SN-38 to the tumor compared to irinotecan, even though 22-fold lessSN-38 equivalents were administered with the ADC.

IMMU-132 Clinical Protocol—

The protocol used in the phase I/II study was as indicated in Table 7below.

TABLE 7 Clinical Protocol Using IMMU-132: OVERVIEW Dosing Once weeklyfor 2 weeks administered every 21 days for up to 8 cycles. regimenPatients with objective responses are allowed to continue beyond 8cycles. In the initial enrollment, the planned dose was delayed andreduced if ≥Grade 2 treatment-related toxicity; protocol was amendedlater in study to dose delay and reduction only in the event of ≥Grade 3toxicity. The development of severe toxicities due to treatment requiresdose reduction by 25% of the assigned dose for 1^(st) occurrence, 50%for 2^(nd) occurrence, and treatment discontinued entirely in the eventof a 3^(rd) occurrence. Dose level 8, 12, 18 mg/kg; later reduced to anintermediate dose level of 10 mg/kg. cohorts Cohort size Standard PhaseI [3 + 3] design; expansion includes 15 patients in select cancers. DLTGrade 4 ANC ≥7 d; ≥Grade 3 febrile neutropenia of any duration; Grade 4Platelets ≥5 d; Grade 4 Hgb; Grade 4 N/V/D of any duration or any Grade3 N/V/D for >48 h; Grade 3 infusion-related reactions; ≥Grade 3 non-heme toxicity at least possibly due to study drug. Maximum Maximum dosewhere ≥2/6 patients tolerate the full 21-d treatment cycle Acceptablewithout dose delay or reduction or ≥Grade 3 toxicity. Dose (MAD)Patients Metastatic colorectal, pancreas, gastric, esophageal, lung(NSCLC, SCLC), triple-negative breast, prostate, ovarian, renal, urinarybladder, head and neck, hepatocellular. Refractory/relapsed afterstandard treatment regimens for metastatic cancer. Prioririnotecan-containing therapy NOT required for enrollment. No bulkylesion >5 cm. Must be 4 weeks beyond any major surgery, and 2 weeksbeyond radiation or chemotherapy regimen. Gilbert's disease or known CNSmetastatic disease are excluded.

Patients were administered IMMU-132 according to the protocol summarizedabove. The response assessment to last prior therapy before IMMU-132treatment is summarized in FIG. 13. The response assessment to IMMU-132administration is shown in FIG. 14. A summary of time to progression(TTP) results following administration of IMMU-132 is shown in FIG. 15.An exemplary case study is as follows. A 34 y/o white male initiallydiagnosed with metastatic pancreatic cancer (liver) had progressed onmultiple chemotherapy regimens, including gemcitabine/Erlotinib/FG-3019,FOLFIRINOX and GTX prior to introduction of IMMU-132 (8 mg/kg dose givendays 1 and 8 of a 21 day cycle). The patient received the drug for 4 mowith good symptomatic tolerance, an improvement in pain, a 72% maximumdecline in CA19-9 (from 15885 U/mL to 4418 U/mL) and stable disease byCT RECIST criteria along with evidence of tumor necrosis. Therapy had tobe suspended due to a liver abscess; the patient expired ˜6 weeks later,6 mo following therapy initiation.

CONCLUSIONS

Preclinical studies indicated that IMMU-132 delivers 120-times theamount of SN-38 to a human pancreatic tumor xenograft than whenirinotecan is given. As part of a larger study enrolling patients withdiverse metastatic solid cancers, the Phase 2 dose of IMMU-132 wasdetermined to be 8 to 10 mg/kg, based on manageable neutropenia anddiarrhea as the major side effects. No anti-antibody or anti-SN-38antibodies have been detected to-date, even with repeated therapeuticcycles.

A study of 14 advanced PDC patients who relapsed after a median of 2prior therapies showed CT-confirmed antitumor activity consisting of8/13 (62%) with stable disease. Median duration of TTP for 13 CTassessable pts was 12.7 weeks compared to 8.0 weeks estimated from lastprior therapy. This ADC, with a known drug of nanomolar toxicity,conjugated to an antibody targeting Trop-2 prevalent on many epithelialcancers, by a linker affording cleavage at the tumor site, represents anew efficacious strategy in pancreatic cancer therapy with ADCs. Incomparison to the present standard of care for pancreatic cancerpatients, the extension of time to progression in pancreatic cancerpatients, particularly in those resistant to multiple prior therapies,was surprising and could not have been predicted.

Example 10. Combining Antibody-Targeted Radiation (Radioimmunotherapy)and Anti-Trop-2-SN-38 ADC Improves Pancreatic Cancer Therapy

We previously reported effective anti-tumor activity in nude micebearing human pancreatic tumors with ⁹⁰Y-humanized PAM4 IgG (hPAM4;⁹⁰Y-clivatuzumab tetraxetan) that was enhanced when combined withgemcitabine (GEM) (Gold et al., Int J. Cancer 109:618-26, 2004; ClinCancer Res 9:3929S-37S, 2003). These studies led to clinical testing offractionated ⁹⁰Y-hPAM4 IgG combined with GEM that is showing encouragingobjective responses. While GEM is known for its radiosensitizingability, alone it is not a very effective therapeutic agent forpancreatic cancer and its dose is limited by hematologic toxicity, whichis also limiting for ⁹⁰Y-hPAM4 IgG.

As discussed in the Examples above, an anti-Trop-2 ADC composed of hRS7IgG linked to SN-38 shows anti-tumor activity in various solid tumors.This ADC is very well tolerated in mice (e.g., ≥60 mg), yet just 4.0 mg(0.5 mg, twice-weekly×4) is significantly therapeutic. Trop-2 is alsoexpressed in most pancreatic cancers.

The present study examined combinations of ⁹⁰Y-hPAM4 IgG with RS7-SN-38in nude mice bearing 0.35 cm³ subcutaneous xenografts of the humanpancreatic cancer cell line, Capan-1. Mice (n=10) were treated with asingle dose of ⁹⁰Y-hPAM4 IgG alone (130 i.e., the maximum tolerated dose(MTD) or 75 with RS7-SN-38 alone (as above), or combinations of the 2agents at the two ⁹⁰Y-hPAM4 dose levels, with the first ADC injectiongiven the same day as the ⁹⁰Y-hPAM4. All treatments were tolerated, with<15% loss in body weight. Objective responses occurred in most animals,but they were more robust in both of the combination groups as comparedto each agent given alone. All animals in the 0.13-mCi ⁹⁰Y-hPAM4 IgG+hRS7-SN-38 group achieved a tumor-free state within 4 weeks, while otheranimals continued to have evidence of persistent disease. These studiesprovide the first evidence that combined radioimmunotherapy and ADCenhances efficacy at safe doses.

In the ongoing PAM4 clinical trials, a four week clinical treatmentcycle is performed. In week 1, subjects are administered a dose of¹¹¹In-hPAM4, followed at least 2 days later by gemcitabine dose. Inweeks 2, 3 and 4, subjects are administered a ⁹⁰Y-hPAM4 dose, followedat least 2 days later by gemcitabine (200 mg/m²). Escalation started at3×6.5 mCi/m². The maximum tolerated dose in front-line pancreatic cancerpatients was 3×15 mCi/m² (hematologic toxicity is dose-limiting). Of 22CT-assessable patients, the disease control rate (CR+PR+SD) was 68%,with 5 (23%) partial responses and 10 (45%) having stabilization as bestresponse by RECIST criteria.

Preparation of Antibody-Drug Conjugate (ADC)

The SN-38 conjugated hRS7 antibody was prepared as described above andaccording to previously described protocols (Moon et al. J Med Chem2008, 51:6916-6926; Govindan et al., Clin Cancer Res 2009.15:6052-6061). A reactive bifunctional derivative of SN-38 (CL2A-SN-38)was prepared. The formula of CL2A-SN-38 is(maleimido-[x]-Lys-PABOCO-20-O-SN-38, where PAB is p-aminobenzyl and ‘x’contains a short PEG). Following reduction of disulfide bonds in theantibody with TCEP, the CL2A-SN-38 was reacted with reduced antibody togenerate the SN-38 conjugated RS7.

⁹⁰Y-hPAM4 is prepared as previously described (Gold et al., Clin CancerRes 2003, 9:3929S-37S; Gold et al., Int J Cancer 2004, 109:618-26).

Combination RAIT+ADC

The Trop-2 antigen is expressed in most epithelial cancers (lung,breast, prostate, ovarian, colorectal, pancreatic) and hRS7-SN-38conjugates are being examined in various human cancer-mouse xenograftmodels. Initial clinical trials with ⁹⁰Y-hPAM4 IgG plus radiosensitizingamounts of GEM are encouraging, with evidence of tumor shrinkage orstable disease. However, therapy of pancreatic cancer is verychallenging. Therefore, a combination therapy was examined to determinewhether it would induce a better response. Specifically, administrationof hRS7-SN-38 at effective, yet non-toxic doses was combined with RAITwith ⁹⁰Y-hPAM4 IgG.

The results demonstrated that the combination of hRS7-SN-38 with⁹⁰Y-hPAM4 was more effective than either treatment alone, or the sum ofthe individual treatments (not shown). At a dosage of 75 μCi ⁹⁰Y-hPAM4,only 1 of 10 mice was tumor-free after 20 weeks of therapy (not shown),the same as observed with hRS7-SN-38 alone (not shown). However, thecombination of hRS7-SN-38 with ⁹⁰Y-hPAM4 resulted in 4 of 10 mice thatwere tumor-free after 20 weeks (not shown), and the remaining subjectsshowed substantial decrease in tumor volume compared with eithertreatment alone (not shown). At 130 μCi ⁹⁰Y-hPAM4 the difference waseven more striking, with 9 of 10 animals tumor-free in the combinedtherapy group compared to 5 of 10 in the RAIT alone group (not shown).These data demonstrate the synergistic effect of the combination ofhRS7-SN-38 with ⁹⁰Y-hPAM4. RAIT+ADC significantly improved time toprogression and increased the frequency of tumor-free treatment. Thecombination of ADC with hRS7-SN-38 added to the MTD of RAIT with⁹⁰Y-hPAM4 had minimal additional toxicity, indicated by the % weightloss of the animal in response to treatment (not shown).

The effect of different sequential treatments on tumor survivalindicated that the optimal effect is obtained when RAIT is administeredfirst, followed by ADC (not shown). In contrast, when ADC isadministered first followed by RAIT, there is a decrease in theincidence of tumor-free animals (not shown). Neither unconjugated hPAM4nor hRS7 antibodies had anti-tumor activity when given alone (notshown).

Example 11. Use of hRS7-SN-38 (IMMU-132) to Treat Therapy-RefractiveMetastatic Breast Cancer

The patient was a 57-year-old woman with stage IV, triple-negative,breast cancer (ER/PR negative, HER-neu negative), originally diagnosedin 2005. She underwent a lumpectomy of her left breast in 2005, followedby Dose-Dense ACT in adjuvant setting in September 2005. She thenreceived radiation therapy, which was completed in November. Localrecurrence of the disease was identified when the patient palpated alump in the contralateral (right) breast in early 2012, and was thentreated with CMF (cyclophosphamide, methotrexate, 5-fluorouracil)chemotherapy. Her disease recurred in the same year, with metastaticlesions in the skin of the chest wall. She then received acarboplatin+TAXOL® chemotherapy regimen, during which thrombocytopeniaresulted. Her disease progressed and she was started on weeklydoxorubicin, which was continued for 6 doses. The skin disease also wasprogressing. An FDG-PET scan on 09/26/12 showed progression of diseaseon the chest wall and enlarged, solid, axillary nodes. The patient wasgiven oxycodone for pain control.

She was given IXEMPRA® from October 2012 until February 2013 (every 2weeks for 4 months), when the chest wall lesion opened up and bled. Shewas then put on XELODA®, which was not tolerated well due to neuropathyin her hands and feet, as well as constipation. The skin lesions wereprogressive and then she was enrolled in the IMMU-132 trial after givinginformed consent. The patient also had a medical history ofhyperthyroidism and visual disturbances, with high risk of CNS disease(however, brain MRI was negative for CNS disease). At the time ofenrollment to this trial, her cutaneous lesions (target) in the rightbreast measured 4.4 cm and 2.0 cm in the largest diameter. She hadanother non-target lesion in the right breast and one enlarged lymphnode each in the right and left axilla.

The first IMMU-132 infusion (12 mg/kg) was started on Mar. 12, 2013,which was tolerated well. Her second infusion was delayed due to Grade 3absolute neutrophil count (ANC) reduction (0.9) on the scheduled day ofinfusion, one week later. After a week delay and after receivingNEULASTA®, her second IMMU-132 was administered, with a 25% dosereduction at 9 mg/kg. Thereafter she has been receiving IMMU-132 onschedule as per protocol, once weekly for 2 weeks, then one week off.Her first response assessment on May 17, 2013, after 3 therapy cycles,showed a 43% decrease in the sum of the long diameter of the targetlesions, constituting a partial response by RECIST criteria. She iscontinuing treatment at the 9 mg/kg dose level. Her overall health andclinical symptoms improved considerably since she started treatment withIMMU-132.

Example 12. Use of hRS7-SN-38 (IMMU-132) to Treat Refractory,Metastatic, Small-Cell Lung Cancer

This is a 65-year-old woman with a diagnosis of small-cell lung cancer,involving her left lung, mediastinal lymph nodes, and Mill evidence of ametastasis to the left parietal brain lobe. Prior chemotherapy includescarboplatin, etoposide, and topotecan, but with no response noted.Radiation therapy also fails to control her disease. She is then givenIMMU-132 at a dose of 18 mg/kg once every three weeks for a total of 5infusions. After the second dose, she experiences hypotension and aGrade 2 neutropenia, which improve before the next infusion. After thefifth infusion, a CT study shows 13% shrinkage of her target left lungmass. MM of the brain also shows a 10% reduction of this metastasis. Shecontinues her IMMU-132 dosing every 3 weeks for another 3 months, andcontinues to show objective and subjective improvement of her condition,with a 25% reduction of the left lung mass and a 21% reduction of thebrain metastasis.

Example 13. Therapy of a Gastric Cancer Patient with Stage IV MetastaticDisease with hRS7-SN-38 (IMMU-132)

This patient is a 60-year-old male with a history of smoking and periodsof excessive alcohol intake over a 40-year-period. He experiences weightloss, eating discomfort and pain not relieved by antacids, frequentabdominal pain, lower back pain, and most recently palpable nodes inboth axilla. He seeks medical advice, and after a workup is shown tohave an adenocarcinoma, including some squamous features, at thegastro-esophageal junction, based on biopsy via a gastroscope.Radiological studies (CT and FDG-PET) also reveal metastatic disease inthe right and left axilla, mediastinal region, lumbar spine, and liver(2 tumors in the right lobe and 1 in the left, all measuring between 2and 4 cm in diameter). His gastric tumor is resected and he is then puton a course of chemotherapy with epirubicin, cisplatin, and5-fluorouracil. After 4 months and a rest period of 6 weeks, he isswitched to docetaxel chemotherapy, which also fails to control hisdisease, based on progression confirmed by CT measurements of themetastatic tumors and some general deterioration.

The patient is then given therapy with IMMU-132 (hRS7-SN-38) at a doseof 10 mg/kg infused every-other-week for a total of 6 doses, after whichCT studies are done to assess status of his disease. These infusions aretolerated well, with some mild nausea and diarrhea, controlled withsymptomatic medications. The CT studies reveal that the sum of his indexmetastatic lesions has decreased by 28%, so he continues on this therapyfor another 5 courses. Follow-up CT studies show that the diseaseremains about 35% reduced by RECIST criteria from his baselinemeasurements prior to IMMU-132 therapy, and his general condition alsoappears to have improved, with the patient regaining an optimisticattitude toward his disease being under control.

Example 14. Clinical Trials of IMMU-132 in Diverse Trop-2 PositiveCancers Abstract

Sacituzumab govitecan (IMMU-132, also known as hRS7-CL2A-SN-38) is anantibody-drug conjugate (ADC) targeting Trop-2, a surface glycoproteinexpressed on many epithelial tumors, for delivery of SN-38, the activemetabolite of irinotecan. Unlike most ADCs that use ultratoxic drugs andstable linkers, IMMU-132 uses a moderately toxic drug with a moderatelystable carbonate bond between SN-38 and the linker. Flow cytometry andimmunohistochemistry disclosed Trop-2 is expressed in a wide range oftumor types, including gastric, pancreatic, triple-negative breast(TNBC), colonic, prostate, and lung. While cell-binding experimentsreveal no significant differences between IMMU-132 and parental hRS7antibody, surface plasmon resonance analysis using a Trop-2 CM5 chipshows a significant binding advantage for IMMU-132 over hRS7. Theconjugate retained binding to the neonatal receptor, but lost greaterthan 60% of the antibody-dependent cell-mediated cytotoxicity activitycompared to hRS7.

Exposure of tumor cells to either free SN-38 or IMMU-132 demonstratedthe same signaling pathways, with pJNK1/2 and p21WAF1/Cip1 up-regulationfollowed by cleavage of caspases 9, 7, and 3, ultimately leading topoly-ADP-ribose polymerase cleavage and double-stranded DNA breaks.Pharmacokinetics of the intact ADC in mice reveals a mean residence time(MRT) of 15.4 h, while the carrier hRS7 antibody cleared at a similarrate as unconjugated antibody (MRT=˜300 h). IMMU-132 treatment of micebearing human gastric cancer xenografts (17.5 mg/kg; twice weekly×4weeks) resulted in significant anti-tumor effects compared to micetreated with a non-specific control. Clinically relevant dosing schemesof IMMU-132 administered either every other week, weekly, or twiceweekly in mice bearing human pancreatic or gastric cancer.

The present Phase I trial evaluated this ADC as a potential therapeuticfor pretreated patients with a variety of metastatic solid cancers. Inparticular embodiments, the therapy is of use to treat patients who hadpreviously been found to be resistant to, or had relapsed from, standardanti-cancer treatments, including but not limited to treatment withirinotecan, the parent compound of SN-38. These results were surprisingand unexpected and could not have been predicted.

Sacituzumab govitecan was administered on days 1 and 8 of 21-day cycles,with cycles repeated until dose-limiting toxicity or progression. Doseescalation followed a standard 3+3 scheme with 4 planned dose levels anddose delay or reduction allowed. Twenty-five patients (52-60 years old,3 median prior chemotherapy regimens) were treated at dose levels of 8(N=7), 10 (N=6), 12 (N=9), and 18 (N=3) mg/kg. Neutropenia wasdose-limiting, with 12 mg/kg the maximum tolerated dose for cycle 1, buttoo toxic with repeated cycles. Lower doses were acceptable for extendedtreatment with no treatment-related grade 4 toxicities and grade 3toxicities limited to fatigue (N=3), neutropenia (N=2), diarrhea (N=1),and leukopenia (N=1). Using CT-based RECIST 1.1 criteria, 3 patientsachieved partial responses (triple-negative breast cancer, small-celllung cancer, colon cancer) and 15 others had stable disease as bestresponse; of these, 12 maintained disease control with continuedtreatment for 16-36 weeks. No pre-selection of patients based on tumorTrop-2 expression was undertaken.

It was concluded that sacituzumab govitecan is a promising ADC conjugatewith acceptable toxicity and encouraging therapeutic activity inpatients with difficult-to-treat cancers. The 8 and 10 mg/kg doses wereselected for Phase II studies.

INTRODUCTION

Two new antibody-drug conjugates (ADCs) incorporating differentultratoxic (picomolar potency) drugs have been approved, leading tofurther development of other ADCs based on similar principles, includinguse of ultratoxic drugs (Younes et al., 2011, Nat Rev Drug Discov11:19-20; Sievers & Senter, 2013, Ann Rev Med 64:15-29; Krop & Winer,2014, Clin Cancer Res 20:15-20). Aternatively, Moon et al. (2008, J MedChem 51:6916-26) and Govindan et al. (2009, Clin Cancer Res 15:6052-61)selected SN-38, a topoisomerase I inhibitor that is the activemetabolite of irinotecan, an approved drug with well-known but complexpharmacology (Mathijssen et al., 2001, Clin Cancer Res 7:2182-94).Several linkers for conjugating SN-38 were evaluated for release fromthe IgG at varying rates, from several hours to days (Moon et al., 2008,J Med Chem 51:6916-26; Govindan et al., 2009, Clin Cancer Res15:6052-61; Cardillo et al., 2011, Clin Cancer Res 17:3157-69). Theoptimal linker that was selected, designated CL2A, exhibiting anintermediate conjugate stability in serum, was attached to the hydroxylgroup on SN-38's lactone ring, thereby protecting this ring from openingto the less toxic carboxylate form while bound to the linker, andcontained a short polyethylene glycol moiety to enhance solubility(Cardillo et al., 2011, Clin Cancer Res 17:3157-69). The active form ofSN-38 was liberated when the carbonate bond between the linker and SN-38was cleaved, which occurred at low pH, such as that found in lysosomes,as well as the tumor microenvironment, or possibly through enzymaticdegradation.

The antibody chosen for this ADC targeted a tumor-associated antigen,Trop-2 (trophoblast cell-surface antigen) (Cardillo et al., 2011, ClinCancer Res 17:3157-69), using the humanized RS7 monoclonal antibody thatwas shown previously to internalize (Stein et al., 1993, Int J Cancer55:938-46. Trop-2 is an important tumor target for an ADC, because it isover-expressed on many epithelial tumors, particularly more aggressivetypes (Ambrogi et al., 2014, PLoS One 9:e96993; Cubas et al., 2009,Biochim Biophys Act 1796:309-14; Trerotola et al., 2013, Oncogene32:222-33). Trop-2 is also present on a number of normal tissues, butpreclinical studies in monkeys that express the antigen observed onlydose-limiting neutropenia and diarrhea with this new ADC, with noevidence of appreciable toxicity to the Trop-2-expressing normal tissues(Cardillo et al., 2011, Clin Cancer Res 17:3157-69). Therefore, withpreclinical data demonstrating activity in several human tumor xenograftmodels and showing a high therapeutic window (Cardillo et al., 2011,Clin Cancer Res 17:3157-69), a Phase I clinical trial was initiated todetermine the maximum tolerated and optimal doses of this novel ADC inheavily-pretreated patients with diverse, relapsed/refractory,metastatic epithelial tumors. This trial was registered atClinicalTrials.gov (NCT01631552).

Materials and Methods

Entry Criteria—

The primary objective was to determine the safety and tolerability ofsacituzumab govitecan (IMMU-132) as a single agent. The trial wasdesigned as a standard 3+3 Phase I design, starting at a dose of 8 mg/kgper injection, with dosages given weekly for 2 weeks in a 3-weektreatment cycle.

Male and non-pregnant, non-lactating females >18 years of age wereeligible if they had a diagnosis of one of thirteen different types ofepithelial tumors. Although no pre-selection based on Trop-2 expressionwas required, these tumors are expected to have Trop-2 expressionin >75% of the cases based on immunohistology studies on archivalspecimens. Patients were required to have measurable metastatic disease(no single lesions >5 cm) and had relapsed or were refractory to atleast one approved standard chemotherapeutic regimen for thatindication. Other key criteria included adequate (grade <1) hematology,liver and renal function, and no known history of anaphylactic reactionsto irinotecan, or grade >3 gastrointestinal toxicity to prior irinotecanor other topoisomerase-I treatments. Since patients with such diversediseases were allowed, prior irinotecan therapy was not a prerequisite.Patients with Gilbert's disease or those who had not toleratedpreviously administered irinotecan or with known CNS metastatic diseasewere excluded.

Study Design—

Baseline evaluations were performed within 4 weeks of the start oftreatment, with regular monitoring of blood counts, serum chemistries,vital signs, and any adverse events. Anti-antibody and anti-SN-38antibody responses were measured by ELISA, with samples taken atbaseline and then prior to the start of every even-numbered treatmentcycle. The first CT examination was obtained 6-8 weeks from the start oftreatment and then continued at 8- to 12-week intervals untilprogression. Additional follow-up was required only to monitor anyongoing treatment-related toxicity. Toxicities were graded using the NCICTCAE version 4.0, and efficacy assessed by RECIST 1.1.

An ELISA to detect Trop-2 in serum was developed that has a sensitivityof 2 ng/mL, but after testing 12 patients and finding no evidence ofcirculating Trop-2, no further screening was performed. Although not aneligibility criterion, specimens of previously archived tumors wererequested for Trop-2 determination by immunohistology, using a goatpolyclonal antibody anti-human Trop-2 (R&D Systems, Minneapolis, Minn.),since the epitope recognized by the ADC's antibody, hRS7, is notpreserved in formalin-fixed, paraffin-embedded sections (Stein et al.,1993, Int J Cancer 55:938-46). Staining was performed as describedbelow.

Therapeutic Regimen—

Lyophilized sacituzumab govitecan was reconstituted in saline andinfused over 2-3 h (100 mg of antibody contained ˜1.6 mg of SN-38, witha mean drug:antibody ratio [DAR] of 7.6:1). Prior to the start of eachinfusion, most patients received acetaminophen, anti-histamines (H1 andH2 blockers), and dexamethasone. Prophylactic use of antiemetics oranti-diarrheal medications was prohibited. Therapy consisted of 2consecutive doses given on days 1 and 8 of a 3-week treatment cycle,with the intent to allow patients to continue treatment for up to 8cycles (i.e., 16 treatments) unless there was unacceptable toxicity orprogression. Patients showing disease stabilization or response after 8cycles could continue treatments.

Dose-limiting toxicities (DLT) were considered as grade >3 febrileneutropenia of any duration, grade 3 thrombocytopenia with significantbleeding or grade 4 thrombocytopenia >5 days, any grade 3 nausea,vomiting or diarrhea that persisted for >48 h despite optimal medicalmanagement, or grade 4 (life threatening) nausea, vomiting or diarrheaof any duration, or any other grade >3 non-hematologic toxicity at leastpossibly due to study drug, as well as the occurrence of any grade 3infusion-related reactions.

The maximum tolerated dose (MTD) was judged on the patient's toleranceto the first treatment cycle. On a scheduled treatment day, any patientwith grade >2 treatment-related toxicity, with the exception ofalopecia, had their treatment delayed in weekly increments for up to 2weeks. Treatment was reinitiated once toxicity had resolved to grade <1.The protocol also initially required all subsequent treatment doses tobe reduced (25% if recovered within 1 week, 50% if within 2 weeks), butthis was relaxed later in the trial when the protocol was amended topermit supportive care after the first cycle. However, if toxicity didnot recover within 3 weeks or worsened, treatment was terminated.Importantly, a dose delay with reduction did not constitute a DLT, andtherefore this allowed treatments to continue, but at a lower dose.Therefore, a patient requiring a dose delay/reduction who was able tocontinue treatment was not considered assessable for DLT, and thenreplaced.

Since a DLT event resulted in the termination of all further treatments,a secondary objective was to assess a dose level that could be toleratedover multiple cycles of treatment with minimal dose delays orreductions. This dose level was designated the maximum acceptable dose,and required patients to tolerate a given dose level in the first cyclewithout having a delay or reduction during that cycle and leading up tothe start of the second cycle.

Pharmacokinetics and Immunogenicity—

Blood samples were taken within ˜30 min from the end of the infusion(e.g., peak) and then prior to each subsequent injection (e.g., trough).Samples were separated and sera frozen for determination of total IgGand sacituzumab govitecan concentrations by ELISA. Serum samples fromseven patients also were assayed for SN-38 content, both total(representing SN-38 bound to the IgG and free) and free SN-38 (i.e.,unbound SN-38).

Results

Patient Characteristics—

Twenty-five patients were enrolled (Table 8). The median age ranged from52 to 60 years, with 76% having an ECOG 1 performance status, theremaining ECOG 0. Most patients had metastatic pancreatic cancer (PDC)(N=7), followed by triple-negative breast cancer (TNBC) (N=4),colorectal cancer (CRC) (N=3), small cell lung cancer (SCLC) (N=2), andgastric cancer (GC) (N=2), with single cases of esophagealadenocarcinoma (EAC), hormone-refractory prostate cancer (HRPC),non-small cell lung cancer (NSCLC), epithelial ovarian cancer (EOC),renal, tonsil, and urinary bladder cancers (UBC).

Immunohistology was performed on archival tissues from 17 patients, with13 (76.4%) having 2+ to 3+ membrane and cytoplasmic staining on >10% ofthe tumor cells in the specimens; 3 specimens (17.6%) were negative.Several representative cases are disclosed below.

All patients entered the trial with metastatic disease in sites typicalfor their primary cancer. CT determined that the median sum of thelargest tumor diameters for all patients was 9.7 cm (range 2.9 to 29.8cm), with 14 patients having 3 or more target lesions (over all patientsmedian=4, range 1-10 lesions) and a median of 2 non-target lesions(range=0-7 lesions) identified in their baseline studies. The mediannumber of prior systemic therapies was 3, with 7 patients (2 PDC and GC,1 each CRC, TNBC, tonsil) having one prior therapy, and 7 having five ormore prior therapies; eleven patients had prior radiation therapy. Priortopoisomerase I therapy was given to nine patients, with 2/3 CRC, 4/7PDC, and 1 patient with EAC receiving irinotecan, and 2/2 patients withSCLC having topotecan, with three of these (2 with SCLC and one withCRC) failing to respond to the anti-topoisomerase 1 therapy. Further,seven of 23 patients (2 undetermined) had responded to their last priortherapy, with a median duration of 3 months (range, 1-11 months).

Nearly all patients received multiple sacituzumab govitecan treatments(median, 10 doses) until there was definitive evidence of diseaseprogression by CT using RECIST 1.1; one patient withdrew becausesystematic deterioration, and 1 patient did not have their target lesionmeasured in first follow-up when a new lesion was observed.

TABLE 8 Baseline demographics and disease characteristics (N = 25patients). 8 mg/kg 10 mg/kg 12 mg/kg 18 mg/kg M/F 2/5 3/3 3/6 2/1 Age, yMedian (range) 52 (43-62) 58.5 (49-80) 60 (50-74) 56 (52-60) ECOGperformance status 0 3 1 2 0 1 1 5 7 3 Tumor Type N N N N Colorectal 2 10 0 Pancreas 3 1 3 0 TNBC 0 1 2 1 SCLC 0 0 1 1 Other^(a) 2 3 3 1 (EOC,GC) (GC, RCC, (UBC, (EAC) Tonsil) NSCLC, HRPC) Trop-2 expression N 1+ 1(TNBC) 2+ 3 (CRC) 3+ 10 (2 each of EAC, PDC, TNBC; 1 each of EOC,Tonsil, NSCLC, SCLC) Negative 3 (1 each of TNBC, Gastric, Renal) Notdetermined 8 (5 PDC, 1 each of HRPC, SCLC, UBC) Prior Therapy N N N NRadiotherapy 2 4 3 2 Systemic therapy^(c) 1 4 2 1 0 2 0 1 3 1 3 1 1 0 14 1 0 2 0 ≥5 1 2 3 1 Prior Topoisomerase I inhibitor 3 1 4 1 Tumormetastases (# patients) N N N N Target and non-target sitesChest/head/neck 0 2 4 3 Liver^(b) 4 (3) 4 (2) 5 (3) 2 (1) Lungs^(b) 4(3) 4 (2) 4 (3) 1 (1) Lymph nodes 3 2 5 2 Abdomen/pelvis 4 3 4 2 Bone 12 2 1 ≥3 target lesions 4 5 5 0 Patients treated 7 6 9 3Delay/adjustment 1^(st) cycle 1 0 5 2 Dose-limiting toxicity 1^(st)cycle 0 0 0 2 # treatments at this dose median (range) 3 (1-31) 10(1-31) 3 (1-8) 1 (1-2) Total # treatments median (range) 6 (3-31) 10(1-31) 12 (4-34) 4 (3-16) ^(a)Other cancers include ovarian (EOC),gastric (GC), urinary bladder (UBC), non-small cell lung cancer (NSCLC),hormone refractory prostate cancer (HRPC), esophageal adenocarcinoma(EAC), renal cell cancer (RCC), and a squamous cell carcinoma of thetonsil. ^(b)Number of patients with liver or lung involvement; inparenthesis number of these patients with both liver and lunginvolvement. ^(b)Systemic therapy includes chemotherapy and other formsof therapy, including biologicals and investigational agents.

Dose Assessment—

There were no dose delays or reductions, nor DLT events in the 3patients (1 CRC, 2 PDC) enrolled at the starting dose level of 8.0mg/kg. At the next dose level of 12 mg/kg, nine patients were enrolledbecause of protocol-required delays in administering the second dosewere encountered. Five patients experienced a delay in the first cycle(4 had a 1-week delay, with 2 given myeloid growth factor support, and 1patient having a 2-week delay before being given a second dose). All but1 of these patients received 12 mg/kg as their second dose. Four of thenine patients at the 12 mg/kg dose level had their third dose thatstarted the second cycle decreased to 9 mg/kg, and the second cycle wasdelayed 1 additional week in 3 patients. Despite these protocol-requireddelays/reductions, none of the 9 patients had a dose-limiting eventduring the first cycle (e.g., 1 patient had disease-related grade 3hemoglobin after first dose, 2 patients with grade 3 neutropenia afterfirst dose were given myeloid growth factors, 1 had grade 3 neutropeniaafter first dose that recovered without support, 2 had grade 3neutropenia after second dose, 2 patients had grade 2 neutropenia afterthe first or second dose, and 1 patient had no adverse events), andtherefore accrual to the 18 mg/kg dose level was allowed. Here, allthree patients had dose delays after their first treatment, with only 1patient receiving the second treatment at 18 mg/kg. Two patients haddose-limiting grade 4 neutropenia, 1 after first dose, the other afterthe second 18 mg/kg dose, with this latter patient also experiencinggrade 2 diarrhea after this dose. Therefore, with 0/9 patients havingDLT in the first cycle at 12 mg/kg, this level was declared the MTD.

Additional dose-finding studies continued to refine the dose level thatwould allow multiple cycles to be given with minimal delay betweentreatments/cycles. Therefore, 4 more patients were enrolled at the 8mg/kg dose level, and a new intermediate level of 10 mg/kg was opened.Of the initial three patients enrolled at 8 mg/kg, two CRC patientscontinued treatment at 8 mg/kg for a total of 31 and 11 treatments,while a PDC patient received three 8 mg/kg doses before dose reductionto 6 mg/kg because of a grade-2 neutropenia on the fourth dose, and thencompleted 3 more treatments at this level before withdrawing due todisease progression. The additional 4 patients received 3 to 9 doses of8 mg/kg before withdrawing with disease progression. Two of thesepatients received only 1 dose before a protocol-required reduction to 6mg/kg, because of a grade-2 rash and grade-2 neutropenia.

Five of the six patients enrolled at 10 mg/kg received 6 to 30 doseswithout reduction before withdrawing due to disease progression. One GCpatient (#9) developed grade 3 febrile neutropenia as well as grade 4hemoglobin after receiving 1 dose. While the febrile neutropenia wasconsidered possibly-related to treatment, because it occurred shortlyafter the first dose, a perforation in the stomach lining was found tolikely contribute to the grade 4 hemoglobin, and was consideredunrelated. Ultimately, the patient had rapid deterioration and died 4weeks from the first dose.

Thus, while the overall results supported 12 mg/kg as the MTD, since 8to 10 mg/kg were better tolerated in the first cycle and permittedrepeated cycles with minimal toxicity, Phase II clinical studies are inprogress to evaluate these 2 dose levels.

Adverse Events—

There were 297 infusion of sacituzumab govitecan given over 2-3 h, withmost investigators electing to pre-medicate prior to each infusion.There were no infusion-related adverse events. While more than half ofthe patients experienced fatigue, nausea, alopecia, diarrhea, andneutropenia that were considered at least likely related to sacituzumabgovitecan treatment; these were mostly grade 1 and 2 (FIG. 16). The mostreported grade 3 or 4 toxicity was neutropenia (N=8), but six of thesepatients were treated initially at 12 and 18 mg/kg. Febrile neutropeniaoccurred in 2 patients, one was the GC patient #9 already mentioned whoreceived only one 10 mg/kg dose, and a second PDC patient (#19), who hadreceived 4 doses of 12 mg/kg. Diarrhea was mild in most patients, withonly three (12%) experiencing grade 3. Two occurred at the 12 mg/kg doselevel, 1 after receiving 4 doses, and the other after the first dose,but this patient received 6 more doses at 12 mg/kg with only grade 2diarrhea reported. Subsequently, both patients were prescribed anover-the-counter anti-diarrheal and treatment continued. There were noother significant toxicities associated with sacituzumab govitecan, buttwo patients reported a grade 2 rash and 3 patients had a grade 1pruritus.

Efficacy—

FIG. 17A provides a graphic representation of the best response asmeasured by the change in target lesions and time-to-progression datafrom patients who had at least one post-treatment CT measurement oftheir target lesions. Four patients with disease progression are notrepresented in the graph, because they did not have a follow-up CTassessment (N=1) or they had new lesions and therefore progressedirrespective of their target lesion status (N=3). Overall, 3 patientshad more than a 30% reduction in their target lesions (partial response,PR). Two of these patients (#3 and #15) had confirmatory follow-up CTs,while the third patient (#22) progressed at the next CT performed 12weeks later. Fifteen patients had stable disease (SD), and 7 progressed(PD) as the best response by RECIST 1.1. The median time to progressionfrom the start of treatment for 24 patients (excluding 1 patient whoreceived only 1 treatment and withdrew) was 3.6 months [range, 1-12.8months]; 4.1 months (range, 2.6-12.8 months) for all patients with SD orPR (N=18). Of the nine patients who received prior therapy containing atopoisomerase-I inhibitor, two had significant reductions of theirtarget lesions (28% and 38%), 5 had stable disease, including 2 forsustained periods (4.1 and 6.9 months, respectively), whereas 2progressed at their first assessment.

FIG. 17B compares TTP with survival of these patients, indicating that16 patients survived from onset of therapy for 15-20 months, includingtwo with a PR (patients 15 (TNBC) and 3 (CRC), and the other four withSD (2 CRC, 1 HRPC, 1 TNBC). Examples of radiological responses in 2patients with >30% reduction in their target lesions (PR) are shown inFIG. 18.

In addition to the 3 patients with PR as best response, there wereseveral notable cases of extended stable disease. A 50-year-old patientwith TNBC (patient 18; immunohistology Trop-2 expression=3+) experienceda 13% reduction after just 3 doses, culminating after 16 doses in a 19%reduction in the 4 target lesions (SLD decreased from 7.5 to 6.1 cm),before progressing 45 weeks after starting treatment and receiving 26doses. A 63-year-old female with CRC (patient 10; immunohistology 2+)with 7 prior treatments, including 3 separate courses of anirinotecan-containing regimens, had an overall 23% reduction in 5 targetlesions after receiving 5 doses of 10 mg/kg sacituzumab govitecan,culminating in a maximum 28% reduction after 18 doses. Her plasma CEAdecreased to 1.6 ng/mL from a baseline level of 38.5 ng/mL. Afterreceiving 25 doses (27 weeks), she had PD with a 20% increase from theSLD nadir. Interesting, plasma CEA at the time treatment ended was only4.5 ng/mL. A 68-year-old patient with HRPC (patient 20; noimmunohistology) presented with 5 target lesions (13.3 cm) and 5non-target lesions (3 bone metastases). He received 34 treatments over aperiod of 12.7 months until progression, with PSA levels increasinggradually over this time. Another notable case was a 52-year-old malewith esophageal cancer (patient 25; immunohistology 3+) who had received6 prior therapies, including 6 months of FOLFIRI as his 3^(rd) course oftreatment. Treatment was initiated at 18 mg/kg of sacituzumab govitecan,which was reduced to 13.5 mg/kg because of neutropenia. He had SD over aperiod of 30 weeks, receiving 15 doses before progressing. A 60-year-oldfemale with PDC (#11) with liver metastases was treated at 10 mg/kg. Herbaseline CA19-9 serum titer decreased from 5880 to 2840 units/mL after 8doses and there was disease stabilization (12% shrinkage as bestresponse) for a period or 15 weeks (11 doses) before a new lesion wasdiscovered. Nevertheless, because CA19-9 remained reduced (2814units/mL), the patient received another 8 treatments (3 months) at 10mg/kg before coming off study with progression of her target lesions.

At this time, the potential utility of testing Trop-2 expression inarchived samples from this small sampling of 16 patients with diversecancers is insufficient to allow for a definitive assessment, primarilybecause most showed elevated expression.

PK and Immunogenicity—

Concentrations of sacituzumab govitecan and IgG in the 30-min serumsample are provided in Table 9, which showed a general trend for thevalues to increase as the dose increased. In a representative case, apatient with TNBC (#15) received multiple doses, starting at 12 mg/kg,with subsequent reductions over the course of her treatment.Concentrations of the IgG and sacituzumab govitecan in the 30-min serumover multiple doses by ELISA were similar over time (not shown),adjusting lower when the dose was reduced. While residual IgG could befound in the serum drawn immediately before the next dose (troughsamples), no sacituzumab govitecan could be detected (not shown).

Total SN-38 concentration in the 30-min serum sample of patient 15 was3,930 ng/mL after the first dose in cycle 1 (C1D1), but when sacituzumabgovitecan treatment was reduced to 9.0 mg/kg for the second dose of thefirst cycle (C1D2), the level decreased to 2,947 ng/mL (not shown). Afurther reduction to 2,381 ng/mL was observed in the 6^(th) cycle, whenthe dose was further reduced to 6.0 mg/kg. The amount of free SN-38 inthese samples ranged from 88 to 102 ng/mL (2.4% to 3.6% of total SN-38),illustrating that >96% of the SN-38 in the serum in these peak sampleswas bound to IgG. Twenty-eight 30-min serum samples from 7 patients wereanalyzed by HPLC, with free SN-38 averaging 2.91±0.91% of the totalSN-38 in these samples. Free SN-38G concentrations measured in 4patients never exceeded SN-38 levels, and were usually several-foldlower. For example, patient #25 had determinations assessed in the30-min sample for 12 injections over 8 cycles of treatment. At astarting dose of 18 mg/kg, he had 5,089 ng/mL of SN-38 in theacid-hydrolyzed sample (total SN-38) and just 155.2 ng/mL in thenon-hydrolyzed sample (free SN-38; 3.0%). Free SN-38G (glucuronidatedform) in this sample was 26.2 ng/mL, or just 14.4% of the total unboundSN-38+SN-38G in the sample. The patient continued treatment at 13.5mg/kg, with SN-38 averaging 3309.8±601.8 ng/mL in the 11 remaining peak,acid-hydrolyzed samples, while free SN-38 averaged 105.4±47.7 ng/mL(i.e., 96.8% bound to the IgG), and free SN-38G averaging 13.9±4.1 ng/mL(11.6% of the total SN-38+SN-38G). Importantly, in nearly all of thepatients, concentrations of SN-38G in the acid-hydrolyzed andnon-hydrolyzed samples were similar, indicating that none of the SN-38bound to the conjugate was glucuronidated.

TABLE 9 Serum concentration (μg/mL) of intact sacituzumab govitecan(ADC) and hRS7 IgG by ELISA. Assays were performed in samples taken 0.5h after the first dose. 8 mg/kg 10 mg/kg 12 mg/kg 18 mg/kg N 7 5 9 3 IgGADC IgG ADC IgG ADC IgG ADC Mean 193.1 141.5 203.35 185.77 239.2 183.3409.16 258.27 SD 56.5 23.8 55.72 54.14 70.7 71.8 88.78 143.26 Min 96.095.0 152.00 144.00 168.9 98.0 321.48 162.80 Max 249.0 169.3 285.85237.39 400.0 311.0 499.00 423.00

None of these patients had a positive baseline level (i.e., >50 ng/mL)or a positive antibody response to either the IgG or SN-38 over theircourse of treatment.

DISCUSSION

Trop-2 is expressed abundantly in many epithelial tumors, making it anantigen of interest for targeted therapies (Cubas et al., 2009, BiochimBiophys Acta 1796:309-14), especially since it is considered aprognostic marker and oncogene in several cancer types (Cardillo et al.,2011, Clin Cancer Res 17:3157-69; Ambrogi et al., 2014, PLoS One9:e96993; Cubas et al., 2009, Biochim Biophys Acta 1796:309-14;Trerotola et al., 2013, Oncogene 32:222-33). Although its expression innormal tissues and relationship to another well-studied tumor-associatedantigen, EpCam, drew some initial words of caution regarding the safetyof developing immunotherapeutics to Trop-2 (Trerotola et al., 2009,Biochim Biophys Acta 1805:119-20), our studies in Cynomolgus monkeysthat express Trop-2 in tissues similar to humans indicated sacituzumabgovitecan was very well tolerated at a human equivalent dose of −40mg/kg (Cardillo et al., 2011, Clin Cancer Res 17:3157-69). At higherdoses, animals experienced neutropenia and diarrhea, known side-effectsassociated with SN-38 derived from irinotecan therapy, yet evidence forsignificant histopathological changes in Trop-2-expressing normaltissues was lacking (Cardillo et al., 2011, Clin Cancer Res 17:3157-69).Thus, with other preclinical studies finding sacituzumab govitecan waspotent at the low nanomolar level and effective in a variety of humanepithelial tumor xenografts at non-toxic doses, a phase I trial wasundertaken in patients who had failed one or more standard therapies fortheir diverse metastatic epithelial tumors.

A major finding of this study was that despite using a more conventionaldrug that is not considered as ultratoxic (drugs active in picomolarrange, whereas SN-38 has potency in the low nanomolar range), thesacituzumab govitecan anti-Trop-2-SN-38 conjugate proved clinically tobe therapeutically active in a wide range of solid cancers at doses withmoderate and manageable toxicity, thus exhibiting a high therapeuticindex. A total of 297 doses of sacituzumab govitecan were given to 25patients without incident; 4 patients received >25 injections.Importantly, no antibody response to the hRS7 IgG or SN-38 was detected,even in patients with multiple cycles of treatment for up to 12 months.Although Trop-2 is expressed in low quantities in a variety of normaltissues (Cardillo et al., 2011, Clin Cancer Res 17:3157-69), neutropeniawas the only dose-limiting toxicity, with myeloid growth factor supportused in 2 patients given >12 mg/kg of sacituzumab govitecan to expediterecovery and allow continuation of treatment in patients who hadexhausted their options for other therapy. While the MTD was declared tobe 12 mg/kg, 8.0 and 10.0 mg/kg dose levels were selected for furtherexpansion, since patients were more likely to tolerate additional cyclesat these levels with minimal supportive care, and responses wereobserved at these levels. Only 2 of 13 patients (15.4%) experiencedgrade-3 neutropenia at these dose levels. The grade 3 and 4 neutropeniaincidence for irinotecan monotherapy given weekly or once every 3 weeksin a front- or second-line setting was 14 to 26% (Camptosar—irinotecanhydrochloride injection, solution (prescribing information, packageinsert) Pfizer, 2012). With sacituzumab govitecan, only 1 patient at the10 mg/kg dose level had grade-3 diarrhea. This incidence is lower thanthe 31% of patients given weekly×4 doses of irinotecan who experiencedgrades 3 and 4 late diarrhea (Camptosar—irinotecan hydrochlorideinjection, solution (prescribing information, package insert) Pfizer,2012). Other common toxicities attributed to sacituzumab govitecanincluded fatigue, nausea, and vomiting, most being grade 1 and 2, aswell as alopecia. Two incidents of febrile neutropenia and one of grade3 deep vein thrombosis also occurred at the 10 and 12 mg/kg dose levels.UGT1A1 monitoring was not initiated until after dose exploration wascompleted, and therefore an assessment of its contribution to toxicitycannot be reported at this time.

Patients enrolled in this trial were not pre-selected for Trop-2expression, primarily because immunohistological assessments of tissuemicroarrays of diverse cancers (such as prostate, breast, pancreas,colorectal, and lung cancers) had indicated the antigen was presentin >90% of the specimens (not shown). In addition, Trop-2 was not foundin the sera of 12 patients with diverse metastatic cancers, furthersuggesting that a serum assay would not be useful for patient selection.Although we are attempting to collect archival specimens of the tumorsfrom patients enrolled in the trial, there is insufficient evidence atthis time to suggest patient selection based on immunohistologicalstaining will correlate with anti-tumor activity, so no patientenrichment based on Trop-2 expression has been undertaken.

As a monotherapy, sacituzumab govitecan had good anti-tumor activity inpatients with diverse metastatic, relapsed/refractory, epithelialtumors, showing appreciable reductions in target lesions by CT, usingRECIST1.1 criteria, including sustained disease stabilization. Three(12%) of the 25 patients (1 each of SCLC [after progressing withtopotecan], TNBC, and colon cancer) had >30% reductions of their targetlesions before progressing 2.9, 4.3, and 7.1 months, respectively, fromthe onset of therapy. Fifteen patients (60%) had SD, with 9 of theseprogressing after >4 months from the start of treatment. Responses ordisease stabilization occurred in 7 of 9 patients who had prior therapywith a topoisomerase I inhibitor-containing drug or regimen. Three ofthese failed to respond to their prior topoisomerase I inhibitor therapy(irinotecan or topotecan), yet sacituzumab govitecan was able to inducetumor shrinkage in 2 of them: 13% in a patient with colon cancer and 38%in the other with SCLC. Thus, sacituzumab govitecan may betherapeutically active in those who failed or relapsed to a priortopoisomerase I-containing regimen, which should be examined further inthe Phase II expansion study.

Although the largest number of patients enrolled in this trial hadadvanced pancreatic ductal cancer (N=7; median time to progression 2.9months]; range, 1.0 to 4.0 months), even in this difficult-to-treatdisease, there have been encouraging reductions in target lesions andCA19-9 serum concentrations to suggest activity (Picozzi et al., 2014,presented at the AACR Special Conference “Pancreatic Cancer: Innovationsin Research and Treatment, New Orleans, La. USA, p. B99). However,responses in patients with TNBC and SCLC are of particular interest,given the need for targeted therapies in these indications. Indeed,additional partial responses in patients with TNBC (Goldenberg et al.,2014, presented at the AACR San Antonio Breast cancer Symposium, SanAntonio, Tex.) and SCLC (Goldenberg et al., 2014, Sci Transl Med)observed in the on-going expansion phase of this trial have suggestedfurther emphasis on these cancers, but encouraging responses in NSCLC,EAC, UBC, and CRC are also being followed. Indeed, in a recent update ofthe on-going trial of sacituzumab govitecan, in 17 TNBC patients studiedto date, an overall response rate (PR) of 29%, with 46% clinical benefitrate (PR+SD>6 months) has been observed. Long-term survival (15-20months) was observed for almost 25% (6/25) of the patients studied, andincluded 2 with PRs and 4 with SD, including patients with TNBC (N=2),CRC (N=3), and HRPC (N=1).

Analysis of the serum samples 30 min after the end of infusionshowed >96% of the SN-38 was bound to the IgG. More detailedpharmacokinetics will be available when the phase II portion of thetrial is completed. HPLC analysis also detected only trace amounts offree SN-38G in the serum, whereas with irinotecan therapy the AUC forthe less active SN-38G is >4.5-fold higher than SN-38 (Xie et al., 2002,J Clin Oncol 20:3293-301). Comparison of SN-38 delivery in tumor-bearinganimals given sacituzumab govitecan and irinotecan has indicated theSN-38 bound to the IgG is not glucuronidated, whereas in animals givenirinotecan, >50% of the total SN-38 in the serum is glucuronidated(Goldenberg et al., 2014, J Clin Oncol 32: Abstract 3107). Moreimportantly, analysis of SN-38 concentrations were ˜135-fold higher inCapan-1 human pancreatic cancer xenografts given sacituzumab govitecanthan irinotecan (Goldenberg et al., 2014, Sci Transl Med). Thus,sacituzumab govitecan has several distinct advantage over non-targetedforms of topoisomerase-I inhibitors: (i) a mechanism that selectivelyretains the conjugate in the tumor (anti-Trop-2 binding), and (ii) thetargeted SN-38 also appears to be fully protected (i.e., notglucuronidated and in the lactone form), such that any SN-38 accreted bythe tumor cells either by the direct internalization of the conjugate orthrough its release into the tumor microenvironment from the conjugatebound to the tumor will be in its most potent form. These resultssuggest that a moderately-toxic, but well understood, cytotoxic agent,SN-38, can be effective as part of a tumor-targeting ADC, such assacituzumab govitecan. But by administering an ADC with amoderately-toxic drug conjugated at a high drug:antibody ratio (7.6:1),higher concentrations of SN-38 can be delivered to the cancers targeted,as suggested in the improved concentration of SN-38 achieved withsacituzumab govitecan compared to that released from irinotecan.

In conclusion, this phase I experience has shown that sacituzumabgovitecan was tolerated with moderate and manageable toxicity, allrelated to the activity of SN-38, with no evidence of damage to normaltissues known to contain Trop-2. Importantly, sacituzumab govitecan wasactive in patients with diverse metastatic solid tumors, even afterfailing prior therapy with topoisomerase-I inhibitors. Thus, it appearsfrom this initial experience that sacituzumab govitecan has a hightherapeutic index, even in patients with tumors not known to beresponsive to topoisomerase I inhibitors, such as SCLC and TNBC. Thisclinical trial is continuing, focusing on starting doses of 8 and 10mg/kg in patients with TNBC, SCLC, and other Trop-2⁺ cancers.

Example 15. Use of IMMU-132 in Triple Negative Breast Cancer (TNBC)

The Trop-2/TACSTD2 gene has been cloned (Fornaro et al., 1995, Int JCancer 62:610-18) and found to encode a transmembrane Ca⁺⁺-signaltransducer (Basu et al., 1995, Int J Cancer 62:472-72; Ripani et al.,1998, Int J Cancer 76:671-76) functionally linked to cell migration andanchorage-independent growth, with higher expression in a variety ofhuman epithelial cancers, including breast, lung, gastric, colorectal,pancreatic, prostatic, cervical, head-and-neck, and ovarian carcinomas,compared to normal tissues (Cardillo et al., 2011, Clin Cancer Res17:3157-69; Stein et al., 1994; Int J Cancer Suppl 8:98-102; Cubas etal., 2009, Biochim Biophys Acta 196:309-14; Trerotola et al., 2013,Oncogene 32:222-33). The increased expression of Trop-2 has beenreported to be necessary and sufficient for stimulation of cancer growth(Trerotola et al., 2013, Oncogene 32:222-33), while a bi-cistroniccyclin D1-Trop-2 mRNA chimera is an oncogene (Guerra et al., 2008,Cancer Res 68:8113-21). Importantly, elevated expression has beenassociated with more aggressive disease and a poor prognosis in severalcancer types (Cubas et al., 2009, Biochim Biophys Acta 196:309-14;Guerra et al., 2008, Cancer Res 68:8113-21; Bignotti et al., 2010, Eur JCancer 46:944-53; Fang et al., 2009, Int J Colorectal Dis 24:875-84;Muhlmann et al., 2009, J Clin Pathol 62:152-58), including breast cancer(Ambrogi et al., 2014, PLoS One 9:e96993; Lin et al., 2013, Exp MolPathol 94:73-8). Increased Trop-2 mRNA is a strong predictor of poorsurvival and lymph node metastasis in patients with invasive ductalbreast cancers, and Kaplan-Meier survival curves showed that breastcancer patients with high Trop-2 expression had a significantly shortersurvival (Lin et al., 2013, Exp Mol Pathol 94:73-8).

Methods

DAR Determination by HIC—

Clinical lots of IMMU-132 were analyzed by hydrophobic interactionchromatography (HIC) using a butyl-NPR HPLC column (Tosoh Bioscience,King of Prussia, Pa.). IMMU-132 injections (100 μg) were resolved with a15-min linear gradient of 2.25-1.5 M NaCl in 25 mM sodium phosphate, pH7.4, run at 1 mL/min and room temperature.

DAR Determination by LC-MS—

Because the interchain disulfides are reduced and the resultingsulfhydryl groups are used for drug conjugation (or blocked), the heavyand light chains resolved during LC-MS analysis without addition ofreducing agents, and were analyzed independently. Different lots ofIMMU-132 were injected on an Agilent 1200 series HPLC using an AerisWidepore C4 reverse-phase HPLC column (3.6 μM, 50×2.1 mm) and resolvedby reverse phase HPLC with a14-min linear gradient of 30-80%acetonitrile in 0.1% formic acid. Electrospray ionization time of flight(ESI-TOF) mass spectrometry was accomplished with an in-line Agilent6210 ESI-TOF mass spectrometer with Vcap, fragmentor and skimmer set to5000V, 300V and 80V, respectively. The entire RP-HPLC peak representingall kappa or heavy chain species were used to generate deconvoluted massspectra.

Cell Lines—

All human cancer cell lines used in this study were purchased from theAmerican Type Culture Collection (Manassas, Va.), except where noted,and all were authenticated by short tandem repeat (STR) assay by theATCC.

Trop-2 Surface Expression on Various Human Breast Carcinoma Cell Lines—

Expression of Trop-2 on the cell surface is based on flow cytometry.Briefly, cells were harvested with Accutase Cell Detachment Solution(Becton Dickinson (BD); Franklin Lakes, N.J.; Cat. No. 561527) andassayed for Trop-2 expression using QuantiBRITE PE beads (BD Cat. No.340495) and a PE-conjugated anti-Trop-2 antibody (eBiosciences, Cat. No.12-6024) following the manufacturer's instructions. Data were acquiredon a FACSCalibur Flow Cytometer (BD) with CellQuest Pro software, withanalysis using Flowjo software (Tree Star; Ashland Oreg.).

In Vitro Cytotoxicity Testing—

Sensitivity to SN-38 was determined using the3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumdye reduction assay (MTS dye reduction assay; Promega, Madison, Wis.).Briefly, cells were plated into 96-well clear, flat-bottomed plates asdescribed above. SN-38 dissolved in DMSO was diluted with media to afinal concentration of 0.004 to 250 nM. Plates were incubated inhumidified chamber for 96 h 37° C./5% CO₂, after which the MTS dye wasadded and placed back into the incubator until untreated control cellshad an absorbance greater than 1.0. Growth inhibition was measured as apercent of growth relative to untreated cells. Dose-response curves weregenerated from the mean of triplicate determinations, and IC₅₀-valueswere calculated using Prism GraphPad Software.

In Vitro Specificity Testing by Flow Cytometry with rH2AX-Stained Cells—

For drug activity testing, HCC1806 and HCC1395 TNBC cell lines cellswere seeded in 6-well plates at 5×10⁵ cells/well and held at 37° C.overnight. After cooling the cells for 10 min on ice, the cells wereincubated with either IMMU-132 or hA20 anti-CD20-SN38 at −20 μg/ml(equal SN38/well for both agents) for 30 minutes on ice, washed threetimes with fresh media, and then returned to 37° C. overnight. Cellswere trypsinized briefly, pelleted by centrifugation, fixed in 4%formalin for 15 min, then washed and permeabilized in 0.15% Triton-X100in PBS for another 15 min. After washing twice with 1% bovine serumalbumin-PBS, cells were incubated with mouse anti-rH2AX-AF488 (EMDMillipore Corporation, Temecula, Calif.) for 45 minutes at 4° C. Thesignal intensity of rH2AX was measured by flow cytometry using a BDFACSCalibur (BD Biosciences, San Jose, Calif.).

IHC of Trop-2 in Tumor Microarrays and Patient Specimens—

This involved standard IHC methods on tissue and microarray sections.Scoring was based on the intensity of the stain in >10% of the tumorcells within the specimen, including negative, 1+(weak), 2+(moderate),and 3+(strong).

In Vivo Therapeutic Studies in Xenograft Models—

SN-38 equivalents in a dose of 250 μg ADC to a 20-gram mouse (12.5mg/kg) is equal to 0.2 mg SN-38/kg. For irinotecan (irinotecan-HClinjection; AREVA Pharmaceuticals, Inc., Elizabethtown, Ky.), 10 mgirinotecan/kg converts to 5.8 mg SN-38/kg based on mass.

Immunoblotting—

Cells (2×10⁶) were plated in 6-well plates overnight. The following daythey were treated with either SN-38 or IMMU-132 at an SN-38concentration equivalent of 0.4 μg/mL (1 μM) for 24 and 48 h. ParentalhRS7 was used as a control for the ADC.

Quantification of SN-38 in Mice with Human Tumor Xenografts—

Two groups, each with 15 animals bearing subcutaneous implants of thehuman pancreatic carcinoma cell line, were administered eitheririnotecan or IMMU-132. At 5 different intervals, 3 animals per groupwere euthanized. The Capan-1 tumors (0.131±0.054 g; N=30) were removedand homogenized in deionized water (DI) (1 part tissue+10 parts DIwater); serum was diluted with an equal part DI water. Serum and tissuehomogenates were extracted and analyzed by reversed-phase HPLC(RP-HPLC). While extracted samples were adequate for detecting productsfrom the irinotecan-treated animals, samples from animals given IMMU-132were split into 2 portions, with one undergoing an acid-hydrolysis stepin order to release all of the SN-38 bound to the IgG, which wouldotherwise go undetected in the extracted samples.

Statistics—

Statistical analyses were performed using GraphPad Prism version 5.00for Windows, GraphPad Software, La Jolla Calif. USA. The specifictesting performed is identified with each study.

Results

SN-18 Structure and Properties—

IMMU-132 utilizes the topoisomerase I inhibitor, SN-38, the watersoluble metabolite of the anticancer camptothecin, irinotecan(7-ethyl-1044-(1-piperidino)-1-piperidino]carbonyloxycamptothecin), thatis therapeutically active in colorectal, lung, cervical, and ovariancancers (Garcia-Carbonero et al., 2002, Clin Cancer Res 8:641061). Animportant advantage for selecting SN-38 is that the drug's in-vivopharmacology is well known. Irinotecan must be cleaved by esterases toform SN-38, which is 2-3 orders of magnitude more potent thanirinotecan, with activity in the low nanomolar range (Kawato et al.,1991, Cancer Res 51:4187-91). At physiological pH, camptothecins existin an equilibrium comprising the more active lactone form and the lessactive (10% potency) open carboxylic acid form (Burke & Mi, 1994, J MedChem 37:40-46).

The design of the SN-38 derivative used in IMMU-132, CL2A-SN-38,addressed multiple challenges in using this drug in the ADC format, andinvolved the following features: (i) A short polyethylene glycol (PEG)moiety was placed in the cross-linker to confer aqueous solubility tothis highly insoluble drug; (ii) a maleimide group was incorporated forfast thiol-maleimide conjugation to mildly reduced antibody, with aspecially-designed synthetic procedure enabling high-yield incorporationof maleimide in the context of assembling the carbonate linkage; (iii) abenzylcarbonate site provided a pH-mediated cleavage site to release thedrug from the linker; and (iv) importantly, the crosslinker was attachedto SN-38's 20-hydroxy position, which kept the lactone ring of the drugfrom opening to the less active carboxylic acid form under physiologicalconditions (Giovanella et al., 2000, Ann NY Acad Sci 922:27-35). Thesynthesis of SN-38 derivatives and the conjugation of CL2A-SN-38 tomildly reduced hRS7 IgG has been described above. The limited reductionprocedure breaks only the interchain disulfide bridges between theheavy-heavy and heavy-light chains, but not the intra-domain disulfides,generating 8 site-specific thiols per antibody molecule. It is thenconjugated to CL2A-SN-38, purified by diafiltration, and lyophilized forstorage. During manufacturing, conditions are adjusted to minimize anyloss of SN-38 from IMMU-132, with the final lyophilized productconsistently having <1% free SN-38 when reconstituted. However, whenplaced in serum and held at 37° C., SN-38 is released from the conjugatewith a half-life of −1 day (not shown).

The release of SN-38 appears to be an important feature of IMMU-132,with this type of linker selected based on efficacy studies that testedSN-38 conjugated to a variety of linkers that had different rates ofSN-38 release, from ˜10 h release half-life to being highly stable (Moonet al., 2008(30, 31). Optimal therapeutic activity was found with aconjugate having an intermediate release rate in serum of −2 days. Wesubsequently improved the manufacturing process for this type of linker,designated CL2A, by removing a phenylalanine residue (Cardillo et al.,2011, Clin Cancer Res 17:3157-64, and then again compared the efficacywith that of another stably-linked anti-Trop-2 conjugate (CL2) that wasdesigned to release SN-38 only under lysosomal conditions (i.e., in thepresence of cathepsin B and pH 5.0). In animal models, the anti-Trop-2conjugate prepared with the CL2A linker yielded better therapeuticresponses than when SN-38 was linked stably, indicating that evenantibodies that internalized quickly benefitted when SN-38 was allowedto be released in serum with a half-life of ˜1 day (Govidan et al. 2013,Mol Cancer Ther 12:968-78). Since clinical studies with radiolabeledantibodies have found the antibodies localize in tumors within a fewhours, reaching peak concentrations within 1 day (Sharkey et al., 1995,Cancer Res 55:5935s-45s), selectively enhanced concentrations of SN-38are delivered locally in the tumor through internalization of the intactconjugate, extracellular release of the free drug, or both mechanisms inconcert.

Drug-Antibody Ratio (DAR) Determination.

Five clinical lots of IMMU-132 were evaluated by hydrophobic interactionHPLC (HIC-HPLC), which resolved three peaks representing species withDARs of 6, 7 and 8, with the greatest fraction comprising a DAR=8 (notshown). IMMU-132 was produced consistently by this manufacturingprocess, with an overall DAR (DARAvE) of 7.60±0.03 among the fiveclinical lots (not shown). HIC-HPLC results were confirmed by liquidchromatography-mass spectrometry (LC-MS) (not shown). The analysisshowed that >99% of the 8 available sulfhydryl groups were coupled withthe CL2A linker, either with or without SN-38. There were nounsubstituted (or N-ethylmaleimide capped) heavy or light chainsdetected. Thus, the difference in DAR among the species results fromSN-38 liberation from the linker during manufacturing and not from alower initial substitution ratio. Once prepared and lyophilized,IMMU-132 has been stable for several years.

Effect of DAR on Pharmacokinetics and Anti-Tumor Efficacy in Mice.

Mice bearing Trop-2⁺ human gastric carcinoma xenografts (NCI-N87) weregiven 2 treatments 7 days apart, each with equal protein (0.5 mg) dosesof IMMU-132 having DARs of 6.89, 3.28, or 1.64 (FIG. 19A). Animalstreated with the ADCs having a DAR of 6.89 had a significantly improvedmedian survival time (MST) compared to mice given ADCs with either 3.38or 1.64 DARs (MST=39 days vs. 25 and 21 days, respectively; P<0.0014).There was no difference between groups treated with the 3.28 or 1.64 DARconjugates and the saline control group.

To further elucidate the importance of a higher DAR, mice bearingNCI-N87 gastric tumors were administered 0.5 mg IMMU-132 with a DAR of6.89 twice weekly for two weeks (FIG. 19B). Another group received twicethe protein (1 mg) dose of an IMMU-132 conjugate with a DAR of 3.28.Although both groups received the same total amount of SN-38 (36 μg)with each dosing scheme, those treated with the 6.89 DAR conjugateinhibited tumor growth significantly more than tumor-bearing animalstreated with the 3.28 DAR conjugate (P=0.0227; AUC). Additionally,treatment with the lower DAR was not significantly different than theuntreated controls. Collectively, these studies indicate that a lowerDAR reduces efficacy.

An examination of the pharmacokinetic behavior of conjugates prepared atthese different ratios was performed in non-tumor-bearing mice given 0.2mg of each conjugate, unconjugated hRS7 IgG, or hRS7 IgG that wasreduced and then capped with N-ethylmaleimide. Serum was taken at 5intervals from 0.5 to 168 h and assayed by ELISA for hRS7 IgG. There wasno significant difference in the clearance of these conjugates comparedto the unconjugated IgG (not shown). Thus, the substitution level didnot affect the pharmacokinetics of the conjugates, and equallyimportant, the reduction of the interchain disulfide bonds did notappear to destabilize the antibody.

Trop-2 Expression in TNBC and SN-38 Sensitivity.

Trop-2 expression was determined by immunohistochemistry (IHC) inseveral tissue microarrays of human tumor specimens. In one microarraycontaining 31 TNBC specimens, as well as 15 hormone-receptor- orHER-2-positive breast cancers, positive staining occurred in over 95% ofthe tumors, with 3+ staining indicated in 65% of the cases.

Table 10 lists 6 human breast cancer cell lines, including four TNBC,showing their surface expression of Trop-2 and sensitivity to SN-38.Trop-2 surface expression in 5 of the 6 cell lines exceeded 90,000copies per cell. SN-38 potency ranged from 2 to 6 nM in 5 of the 6 celllines, with MCF-7 having the lowest sensitivity of 33 nM. In vitropotency for IMMU-132 is not provided, because nearly all of the SN-38associated with IMMU-132 is released into the media during the 4-dayincubation period, and therefore its potency would be similar to that ofSN-38. Therefore, a different strategy was required to illustrate theimportance of antibody targeting as a mechanism for delivering SN-38.

TABLE 10 Trop-2 expression and SN-38 sensitivity in breast cancer celllines. Receptor Trop-2 surface IC₅₀ (nM) Cell Line status expression^(A)SN-38 SK-BR-3 HER2⁺ 328,281 ± 47,996 2 MDA-MB-468 TNBC 301,603 ± 29,4702 HCC38 TNBC 181,488 ± 69,351 2 MCF-7 ER⁺ 110,646 ± 17,233 33 HCC1806TNBC  91,403 ± 20,817 1 MDA-MB-231 TNBC 32,380 ± 5,460 6 ^(A)Mean ± SDnumber of surface Trop-2 molecules per cell from three separate assays.

Antigen-positive (HCC1806) or -negative (HCC1395) TNBC cell lines thatwere incubated at 4° C. for 30 min with either IMMU-132 or a non-bindinganti-CD20 SN-38 conjugate. The cells were then washed to remove unboundconjugate, and then incubated overnight at 37° C. Cells were fixed andpermeabilized, and then stained with the fluorescentanti-phospho-histone H2A.X antibody to detect dsDNA breaks by flowcytometry (Bonner et al., 2008, Nat Rev Cancer 8:957-67) (Table 11). TheTrop-2⁺ breast cancer cell line, HCC1806, when incubated with IMMU-132,had an increase in median fluorescence intensity (MFI) from 168(untreated baseline) to 546, indicating the increased presence of dsDNAbreaks, whereas the MFI for cells incubated with the non-bindingconjugate remained at baseline levels. In contrast, MFI for the Trop-2antigen-negative cell line, HCC1395, remained at baseline levelsfollowing treatment with either IMMU-132 or the non-binding controlconjugate. Thus, the specificity of IMMU-132 over an irrelevant ADC wasconclusively revealed by evidence of dsDNA breaks only inTrop-2-expressing cells incubated with the anti-Trop-2-bindingconjugate.

TABLE 11 Specificity of IMMU-132 anti-tumor activity in vitro using flowcytometry with phospho-H2AX (anti-histone)-stained cells.^(A) Medianfluorescence intensity HCC1806 HCC1395 Treatment (Trop-2⁺) (Trop-2⁻)Cell alone 4.25 5.54 Cell + anti-rH2AX-AF488 168 122 Cell + IMMU-132 +anti-rH2AX-AF488 546 123 Cell + hA20-SN38 + anti-rH2AX-AF488 167 123^(A)HCC1806 (Trop-2⁺) or HCC1395 (Trop-2⁻) were incubated at 4° C. withIMMU-132 or a non-binding control conjugate (anti-CD20-SN-38) for 30min, washed and incubated overnight at 37° C. in fresh drug-free media.Cells were harvested, fixed, and permeabilized, then stained with thefluorescently-conjugated anti-histone antibody (rH2AX-AF488) fordetection of double-stranded DNA breaks. The median fluorescenceintensity (MFI) is given for (a) background staining of the cells alone(no anti-histone antibody), (b) the background level of dsDNA breaks forthe cells that had no prior exposure to the conjugates, and (c) afterexposed to IMMU-132 or hA20-SN-38 conjugates.

In Vivo Efficacy of hRS7-CL2A-SN-38 in TNBC Xenografts.

The efficacy of IMMU-132 was assessed in nude mice bearing MDA-MB-468TNBC tumors (FIG. 20A). IMMU-132 at a dose of 0.12 or 0.20 mg/kgSN-38-equivalents (0.15 and 0.25 mg IMMU-132/dose) induced significanttumor regression, compared to saline, irinotecan (10 mg/kg; ˜5.8 mg/kgSN-38 equivalents by weight), or a control anti-CD20 ADC,hA20-CL2A-SN-38, given at the same 2 dose levels (P<0.0017, area underthe curve, AUC). Since mice convert irinotecan to SN-38 more efficientlythan humans (38) (in our studies, it averaged ˜25%, see below), at thisirinotecan dose ˜145 to 174 μg of SN-38 would be produced, while theadministered dose of IMMU-132 contained only 9.6 μg. Nevertheless,because IMMU-132 selectively targeted SN-38 to the tumors, it was moreefficacious. These results corroborate findings in other solid tumormodels (Cardillo et al., 2011, Clin Cancer Res 17:3157-69) showing thatspecific targeting of a small amount of SN-38 to the tumor with IMMU-132is much more effective than a much larger dose of irinotecan, or forthat matter a mixture of hRS7 IgG with an equal amount of free SN-38(Cardillo et al., 2011, Clin Cancer Res 17:3157-69). The unconjugatedRS7 antibody, even at repeated doses of 1 mg per animal, did not showany antitumor effects (Cardillo et al., 2011, Clin Cancer Res17:3157-69). However, in-vitro studies with gynecological cancersexpressing Trop-2 have indicated cell killing with the RS7 mAb byantibody-dependent cellular cytotoxicity (Bignotti et al., 2010, Eur JCancer 46:944-53; Raji et al., 2011, J Exp Clin Cancer Res 30:106;Varughese et al., 2011, Gynecol Oncol 122:171-7; Varughese et al., 2011,Am J Obstet Gyneol 205:567). Also, a monovalent Fab of anotheranti-Trop-2 antibody has been reported to be therapeutically active invitro and in animal studies.

On therapy day 56, four of the seven tumors in mice given 0.12 mg/kg ofthe hA20-CL2A-SN-38 control ADC already had progressed to the endpointof 1.0 cm³ (FIG. 20B). At this time, these animals were treated withIMMU-132, electing to use the higher dose of 0.2 mg/kg in an attempt toaffect the progression of these much larger tumors. Despite thesubstantial size of the tumors in several animals, all mice demonstrateda therapeutic response, with tumors significantly smaller in size fiveweeks later (total volume [TV]=0.14±0.14 cm³ vs. 0.74±0.41 cm³,respectively; P=0.0031, two-tailed t-test). Similarly, we chose twoanimals in the irinotecan-treated group with tumors that progressed to˜0.7 cm³ and re-treated one with irinotecan and the other with IMMU-132(not shown). Within 2 weeks of ending treatment, the tumor in theirinotecan-treated animal decreased 23% and then began to progress,while the tumor treated with IMMU-132 had stabilized with a 60% decreasein tumor size. These results demonstrate that even in tumors thatcontinued to grow after exposure to SN-38 via a non-specific ADC, asignificantly enhanced therapeutic response could be achieved whentreated with the Trop-2-specific IMMU-132. However, specific therapeuticeffects with IMMU-132 were not achieved in MDA-MB-231 (FIG. 20C). Thiscell line had the lowest Trop-2 levels, but also was the least sensitiveto SN-38.

Mechanism of Action of IMMU-132 in TNBC—

The apoptotic pathway utilized by IMMU-132 was examined in the TNBC cellline, MDA-MB-468, and in the HER2^(−/−) SK-BR-3 cell line, in order toconfirm that the ADC functions on the basis of its incorporated SN-38(not shown). SN-38 alone and IMMU-132 mediated >2-fold up-regulation ofp21^(WAF1/Cip1) within 24 h in MDA-MB-468, and by 48 h, the amount ofp21^(WAF1/Cip1) in these cells began to decrease (31% and 43% with SN-38or IMMU-132, respectively). Interestingly, in the HER2^(−/−) SK-BR-3tumor line, neither SN-38 nor IMMU-132 mediated the up-regulation ofp21^(WAF1/Cip1) above constitutive levels in the first 24 h, but as seenin MDA-MB-468 cells after 48-h exposure to SN-38 or IMIMU-132, theamount of p21^(WAF1/Cip1) decreased >57%. Both SN-38 and IMMU-132resulted in cleavage of pro-caspase-3 into its active fragments within24 h, but with the greater degree of active fragments observed afterexposure for 48 h. Of note, in both cell lines, IMIMU-132 mediated agreater degree of pro-caspase-3 cleavage, with the highest levelobserved after 48 h when compared to cells exposed to SN-38. Finally,SN-38 and IMMU-132 both mediated poly ADP ribose polymerase (PARP)cleavage, starting at 24 h, with near complete cleavage after 48 h.Taken together, these results confirm that IMMU-132 has a mechanism ofaction similar to that of free SN-38 when administered in vitro.

Delivery of SN-38 by IMMU-132 vs. Irinotecan in a Human Tumor XenograftModel—

Constitutive products derived from irinotecan or IMIMU-132 weredetermined in the serum and tumors of mice implanted s.c. with a humanpancreatic cancer xenograft (Capan-1) administered irinotecan (773 μg;SN-38 equivalents=448 μg) and IMMU-132 (1.0 mg; SN-38 equivalents=16μg).

Irinotecan cleared very rapidly from serum, with conversion to SN-38 andSN-38G seen within 5 min. None of the products was detected at 24 h. TheAUCs over a 6-h period were 21.0, 2.5, and 2.8 μg/mL·h for irinotecan,SN-38, and SN-38G, respectively (SN-38 conversion inmice=[2.5+2.8)/21=25.2%]). Animals given IMMU-132 had much lowerconcentrations of free SN-38 in the serum, but it was detected through48 h (FIG. 5A). Free SN-38G was detected only at 1 and 6 h, and was 3-to 7-times lower than free SN-38.

In the Capan-1 tumors excised from irinotecan-treated animals,irinotecan levels were high over 6 h, but undetectable a 24 h(AUC_(5min-6h)=48.4 μg/g·h). SN-38 was much lower and detected onlythrough 2 h (i.e., AUC_(5min-2h)=0.4 μg/g·h), with SN-38G values almost3-fold higher (AUC=1.1 μg/g·h) (not shown). Tumors taken from animalsgiven IMMU-132 did not have any detectable free SN-38 or SN-38G, butinstead, all SN-38 in the tumor was bound to IMMU-132. Importantly,since no SN-38G was detected in the tumors, this suggests SN-38 bound toIMMU-132 was not glucuronidated. The AUC for SN-38 bound to IMMU-132 inthese tumors was 54.3 μg/g·h, which is 135-fold higher than the amountof SN-38 in the tumors of animals treated with irinotecan over the 2-hperiod that SN-38 could be detected, even though mice given irinotecanreceived 28-fold more SN-38 equivalents than administered with IMIMU-132(i.e., 448 vs 16 μg SN-38 equivalents, respectively)

DISCUSSION

We describe a new ADC targeting Trop-2, and early clinical resultssuggest it is well tolerated and effective in patients with TNBC, aswell as other Trop-2⁺ cancers (Bardia et al., 2014, San Antonio BreastCancer Symposium, P5-19-27). Due to its distinct properties, IMMU-132represents a second-generation ADC. Typically, ADCs require 4 broadattributes to be optimally-effective: (i) selective targeting/activity;(ii) binding, affinity, internalization, and immunogenicity of theantibody used in the ADC; (iii) the drug, its potency, metabolism andpharmacological disposition, and (iv) how the drug is bound to theantibody. Target selectivity is the most common requirement for allADCs, since this will play a major role in defining the therapeuticindex (ratio of toxicity to tumor vs. normal cells). Trop-2 appears tohave both a high prevalence on a number of epithelial cancers, but it isalso expressed by several normal tissues (Cubas et al., 2009, BiochimBiophys Acta 1796:309-14; Trerotola et al., 2013, Oncogene 32:222-33;Stepan et al., 2011, 59:701-10), which could have impacted specificity.However, expression in normal tissues appears to be lower than incancers (Bignotti et al., 2010, Eur J Cancer 46:944-53), and Trop-2appears to be shielded by normal tissue architecture that limitsaccessibility to an antibody, whereas in cancer, these tissue barriersare compromised by the invading tumor. Evidence of this was apparentfrom initial toxicological studies in monkeys, where despite escalatingIMMU-132 doses to levels leading to irinotecan-like neutropenia anddiarrhea, histopathological damage to Trop-2-expressing normal tissuesdid not occur (Cardillo et al., 2011, Clin Cancer Res 17:3157-69). Theseresults appear to have been confirmed clinically, where no specificorgan toxicity has been noted in patients to-date, except for the knowntoxicities of the parental compound, irinotecan (Bardia et al., 2014,San Antonio Breast Cancer Symposium, P5-19-27), which are moremanageable with IMMU-132.

A generally accepted and important criterion for ADC therapy is that theantibody should internalize, delivering its chemotherapeutic inside thecell, where it is usually metabolized in lysosomes. Despite IMMU-132'sinternalization, we believe that the linker in this ADC, which affordslocal release of SN-38 that likely can induce a bystander effect on thecancer cells, is another feature that sets this platform apart fromthose using an ultratoxic drug. Indeed, having an ultratoxic agentlinked stably to the IgG is the only configuration that would preserve auseful therapeutic window for those types of compounds. However, using amore moderately-toxic drug does not give the latitude to use a linkerthat would release the drug too early once in the circulation. Our groupexplored linkers that released SN-38 from the conjugate with differenthalf-lives in serum, ranging from ˜10 h to a highly stable linker, butit was the linker with the intermediate stability that provided the besttherapeutic response in mouse-human tumor xenograft models (Moon et al.,2008, J Med Chem 51:6916-26; Govindan et al., 2009, Clin Chem Res15:6052-61). Since this initial work, we showed that a highly stablelinkage of SN-38 was significantly less effective than the CL2A linkerthat has a more intermediate stability in serum (Govindan et al., 2013,Mol Cancer Ther 12:968-78).

Another current tenet of ADC design is to use an ultra-cytotoxic drug tocompensate for low levels of antibody accretion in tumors, typically0.003 to 0.08% of the injected dose per gram (Sharkey et al., 1995,Cancer Res 55:5935s-45s). The current generation of ultratoxic-drugconjugates have found a drug:antibody substitutions of <4:1 to beoptimal, since higher ratios adversely affected their pharmacokineticsand diminished the therapeutic index by collateral toxicities (Hamblettet al., 2004, Clin Cancer Res 10:7063-70). In this second-generation ADCplatform, we elected to use an IgG-coupling method thatsite-specifically links the drug to the interchain disulfides throughmild reduction of the IgG, which exposes 8 binding sites. With theCL2A-SN-38 linker, we achieved a DAR of 7.6:1, with LC-MS data showingeach of the 8 coupling sites bears the CL2A linker, but apparently someSN-38 is lost during the manufacturing procedure. Nevertheless, 95% ofthe CL2A linker has 7-8 SN-38 molecules. We found subsequently that (a)coupling to these sites does not destabilize the antibody, and (b)conjugates prepared with these sites substituted at higher levels didnot compromise antibody binding, nor did it affect pharmacokineticproperties. Indeed, we demonstrated that conjugates prepared at themaximum substitution level had the best therapeutic response inmouse-human tumor xenograft models.

One of the more notable features of IMMU-132 from a tolerabilityperspective is that the SN-38 bound to IgG is not glucuronidated, whichis a critical step in the detoxification of irinotecan. With irinotecantherapy, most of the SN-38 generated is readily converted in the liverto the inactive SN-38G form. Estimates of the AUC for SN-38G show it isoften 4.5- to 32-times higher than SN-38 (Gupta et al., 1994, Cancer Res54:3723-25; Xie et al., 2002, J Clin Oncol 20:3293-301). SN-38G'ssecretion into the bile and subsequent deconjugation bybeta-glucuronidase produced by the intestinal flora is stronglyimplicated in the enterohepatic recirculation of SN-38 and the delayedsevere diarrhea observed with irinotecan (Stein et al., 2010, Ther AdvMed Oncol 2:51-63). After IMMU-132 administration, concentrations ofSN-38G were very low in our animal and clinical studies (e.g., in theserum of patients given IMU-132, only 20-40% of the free SN-38 levelsare in the form of SN-38G), providing strong evidence that SN-38 boundto IgG is largely protected from glucuronidation, even though the10-hydroxy position of the SN-38 is available. We speculate that lowlevels of SN-38G generated by IMMU-132 contributes to the lowerincidence and intensity of diarrhea in patients receiving this ADCcompared to irinotecan therapy.

Preventing glucuronidation of the SN-38 bound to the antibody may alsocontribute to improved therapeutic effects for SN-38 delivered to thetumor. Extracts of tumors from animals given irinotecan found highlevels of irinotecan, with 10-fold lower concentrations of SN-38 andSN-38G. In contrast, the only SN-38 found in the tumors of animals givenIMMU-132 was SN-38 bound to the IgG. We hypothesize that the conjugateretained in the tumor will eventually be internalized, thereby releasingits SN-38 payload, or SN-38 could be release outside the tumor cell;however, it would be released in its fully active form, with a lowerlikelihood of being converted to SN-38G, which occurs primarily in theliver. It is also important to emphasize that by coupling the linker tothe 20-hydroxy position of SN-38, the SN-38 is maintained in the activelactone form (Zhao et al., 2000, J Org Chem 65:4601-6). Collectively,these results suggest that IMMU-132 is able to deliver and concentrateSN-38 to Trop-2⁺ tumors in a selective manner compared to SN-38 derivedfrom non-targeted irinotecan, with the SN-38 delivered by IMMU-132likely being released in the tumor in the fully active,non-glucuronidated, lactone form.

Irinotecan is not conventionally used to treat breast cancer patients.However, the experiments shown here with TNBC cell lines indicate thatconcentrating higher amounts of SN-38 into the tumor enhances itsactivity. In both the MDA-MB-468 TNBC and HER2^(−/−) SK-BR-3 tumorlines, IMMU-132 mediated the activation of the intrinsic apoptoticpathway, with cleavage of pro-caspases into their active fragments andPARP cleavage. The demonstration of double-stranded DNA breaks of cancercells treated with IMMU-132 (Bardia et al., 2014, San Antonio BreastCancer Symposium, P5-19-27) compared to an irrelevant SN-38 ADC)confirms the selective delivery of SN-38 into the target cells. Mostimportantly, these laboratory findings are confirmed by therapy ofpatients with heavily-pretreated, metastatic TNBC, where durableobjective responses have been observed (Bardia et al., 2014, San AntonioBreast Cancer Symposium, P5-19-27). It also appears that IMMU-132 isactive in patients with other cancers and who have failed a priortherapy regimen containing a topoisomerase I inhibitor (Starodub et al.,2015, Clin Cancer Res 21:3870-78).

In conclusion, the use of SN-38 conjugated at a very high ratio of drugto antibody, using a moderately-stable linker, is efficacious in animalmodels and also clinically, constituting a second-generation ADCplatform. Our findings indicate that Trop-2 is a clinically-relevant andnovel target in Trop-2+ solid tumors, particularly TNBC.

Example 16. Studies on the Mechanism of Action of IMMU-132

Sacituzumab govitecan (IMMU-132, also known as hRS7-CL2A-SN-38) is anantibody-drug conjugate (ADC) targeting Trop-2, a surface glycoproteinexpressed on many epithelial tumors, for delivery of SN-38, the activemetabolite of irinotecan. Unlike most ADCs that use ultratoxic drugs andstable linkers, IMMU-132 uses a moderately toxic drug with a moderatelystable carbonate bond between SN-38 and the linker. Flow cytometry andimmunohistochemistry disclosed Trop-2 is expressed in a wide range oftumor types, including gastric, pancreatic, triple-negative breast(TNBC), colonic, prostate, and lung. While cell-binding experimentsreveal no significant differences between IMMU-132 and parental hRS7antibody, surface plasmon resonance analysis using a Trop-2 CM5 chipshows a significant binding advantage for IMMU-132 over hRS7. Theconjugate retained binding to the neonatal receptor, but lost greaterthan 60% of the antibody-dependent cell-mediated cytotoxicity activitycompared to hRS7.

Exposure of tumor cells to either free SN-38 or IMMU-132 demonstratedthe same signaling pathways, with pJNK1/2 and p21WAF1/Cip1 up-regulationfollowed by cleavage of caspases 9, 7, and 3, ultimately leading topoly-ADP-ribose polymerase cleavage and double-stranded DNA breaks.

Pharmacokinetics of the intact ADC in mice reveals a mean residence time(MRT) of 15.4 h, while the carrier hRS7 antibody cleared at a similarrate as unconjugated antibody (MRT=˜300 h). IMMU-132 treatment of micebearing human gastric cancer xenografts (17.5 mg/kg; twice weekly×4weeks) resulted in significant anti-tumor effects compared to micetreated with a non-specific control. Clinically relevant dosing schemesof IMMU-132 administered either every other week, weekly, or twiceweekly in mice bearing human pancreatic or gastric cancer xenograftsdemonstrate similar, significant anti-tumor effects in both models.Current Phase I/II clinical trials (ClinicalTrials.gov, NCT01631552)confirm anticancer activity of IMMU-132 in cancers expressing Trop-2,including gastric and pancreatic cancer patients.

INTRODUCTION

There will be an estimated 22,220 new cases of gastric cancer diagnosedin the United States this year, with a further 10,990 deaths attributedto this disease (Siegel et al., 2014, CA Cancer J Clin 64:9-29). While5-year survival rates are trending upward (currently at 29%), they arestill quite low when compared to most others, including cancers of thecolon, breast, and prostate (65%, 90%, and 100%, respectively). In fact,among human cancers, only esophageal, liver, lung, and pancreatic haveworse 5-year survival rates. Pancreatic cancer remains the fourthleading cause of all cancer deaths in the U.S., with a 5-year survivalrate of only 6% (Siegel et al., 2014, CA Cancer J Clin 64:9-29). It isclear from such grim statistics for gastric and pancreatic cancer thatnew therapeutic approaches are needed.

Trop-2 is a 45-kDa glycoprotein that belongs to the TACSTD gene family,specifically TACSTD22. Overexpression of this trans-membrane protein onmany different epithelial cancers has been linked to an overall poorprognosis. Trop-2 is essential for anchorage-independent cell growth andtumorigenesis (Wang et al., 2008, Mol Cancer Ther 7:280-85; Trerotola etal., 2013, Oncogene 32:222-33). It functions as a calcium signaltransducer that requires an intact cytoplasmic tail that isphosphorylated by protein kinase C12-14. Pro-growth signaling associatedwith Trop-2 includes NF-κB, cyclin D1 and ERK (Guerra et al., 2013,Oncogene 32:1594-1600; Cubas et al., 2010, Mol Cancer 9:253).

In pancreatic cancer, Trop-2 overexpression was observed in 55% ofpatients studied, with a positive correlation with metastasis, tumorgrade, and poor progression-free survival of patients who underwentsurgery with curative intent (Fong et al., 2008, Br J Cancer99:1290-95). Likewise, in gastric cancer 56% of patients exhibitedTrop-2 overexpression on their tumors, which again correlated withshorter disease-free survival and a poorer prognosis in those patientswith lymph node involvement of Trop-2-positive tumor cells (Muhlmann etal., 2009, J Clin Pathol 63:152-58). Given these characteristics and thefact that Trop-2 is linked to so many intractable cancers, Trop-2 is anattractive target for therapeutic intervention with an antibody-drugconjugate (ADC).

A general paradigm for using an antibody to target a drug to a tumorincludes several key features, among them: (a) an antigen target that ispreferentially expressed on the tumor versus normal tissue, (b) anantibody that has good affinity and is internalized by the tumor cell,and (c) an ultra-toxic drug that is coupled stably to the antibody(Panowski et al., 2014, mAbs 6:34-45). Along these lines, we developedan antibody, designated RS7-3G11 (RS7), that bound to Trop-2 in a numberof solid tumors (Stein et al., 1993, Int J Cancer 55:938-46; Basu etal., 1995, Int J Cancer 62:472-79) with nanomolar affinity (Cardillo etal., 2011k, Clin Cancer Res 17:3157-69), and once bound to Trop-2, isinternalized by the cell (Shih et al., 1995, Cancer Res 55:5857s-63s).

By immunohistochemistry, Trop-2 is expressed in some normal tissues,though usually at much lower intensities when compared to neoplastictissue, and often is present in regions of the tissues with restrictedvascular access (Trerotola et al., 2013, Oncogene 32:222-33). Based onthese characteristics, RS7 was humanized and conjugated with the activemetabolite of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38). Invitro cytotoxicity in numerous cell lines has found IC₅₀-values in thesingle digit nanomolar range for SN-38, compared to picomolar range formany of the ultra-toxic drugs currently used in ADCs (Cardillo et al.,2011, Clin Cancer Res 17:3157-69). While the prevailing opinion is touse ultra-toxic agents, such as auristatins or maytansines, to make ADCswith only 2-4 drugs per antibody linked stably to the antibody, suchagents have a narrow therapeutic window, resulting in renewed efforts tore-engineer ADCs to broaden their therapeutic index (Junutula et al.,2010, Clin Cancer Res 16:4769-78).

As one approach to diverge from this practice, we conjugated 7-8 SN-38molecules per antibody using a linker that releases SN-38 with half-lifeof ˜1 day in human serum. It is hypothesized that using a less stablelinker allows for SN-38 release at the tumor site after the ADC targetsthe cells, making the drug accessible to surrounding tumor cells and notjust cells directly targeted by the ADC. The resulting ADC,hRS7-CL2A-SN-38 (sacituzumab govitecan, or IMMU-132), has shownanti-tumor activity against a wide range of tumor types (Cardillo etal., 2011, Clin Cancer Res 17:3157-69). More recently, IMMU-132 hasdemonstrated significant anti-tumor activity against a pre-clinicalmodel of triple negative breast cancer (TNBC) (Goldenberg et al., 2014,Poster presented at San Antonia Breast Cancer Symposium, December 9-13,Abstr. P5-19-08). Most importantly, in a current Phase I/II clinicaltrial, IMMU-132 has shown activity in TNBC patients (Bardia et al.,2014, Poster presented at San Antonia Breast Cancer Symposium, December9-13, Abstr. P5-19-2), thus validating this paradigm shift in ADCchemistry using a less toxic drug and a linker that releases SN-38 overtime rather than being totally dependent on internalization of the ADCto achieve activity.

SN-38 is a known topoisomerase-I inhibitor that induces significantdamage to a cell's DNA. It mediates the up-regulation of earlypro-apoptotic proteins, p53 and p21WAF1/Cip1, resulting in caspaseactivation and poly-ADP-ribose polymerase (PARP) cleavage. Expression ofp21WAF1/Cip1 is associated with G1 arrest of the cell cycle and is thusa hallmark of the intrinsic apoptotic pathway. We demonstratedpreviously that IMMU-132 likewise could mediate the up-regulation ofearly pro-apoptosis signaling events (p53 and p21WAF1/Cip1) resulting inPARP cleavage in NSCLC (Calu-3) and pancreatic (BxPC-3) cell linesconsistent with the intrinsic pro-apoptosis signaling pathway (Cardilloet al., 2011, Clin Cancer Res 17:3157-69).

Herein, we further characterize IMMU-132, with particular attentiontowards the treatment of solid cancers, especially human gastric andpancreatic tumors. Trop-2 surface expression across a range of solidtumor types is examined and correlated with in vivo expression in tumorxenografts. Mechanistic studies further elucidate the intrinsicpro-apoptotic signaling events mediated by IMMU-132, including evidenceof increased double stranded DNA (dsDNA) breaks and later caspaseactivation. Finally, clinically-relevant and non-toxic dosing schemesare compared in gastric and pancreatic carcinoma disease models, testingtwice-weekly, weekly, and every other week schedules to ascertain whichtreatment cycle may be best applied to a clinical setting without lossof efficacy.

Experimental Procedures

Cell Lines and Chemotherapeutics—

All human cancer cell lines used were purchased from the American TypeCulture Collection (ATCC) (Manassas, Va.). Each was maintained accordingto the recommendations of ATCC and routinely tested for mycoplasma, andall were authenticated by short tandem repeat (STR) assay by the ATCC.IMMU-132 (hRS7-SN-38) and control ADCs (anti-CD20 hA20-SN-38 andanti-CD22 hLL2-SN-38) were made as previously described and stored at−20° C. (Cardillo et al., 2011, Clin Cancer Res 17:3157-69). SN-38 waspurchased (Biddle Sawyer Pharma, LLC, New York, N.Y.) and stored in 1 mMaliquots in DMSO at −20° C.

Trop-2 ELISA—

Recombinant human Trop-2 with a His-tag (Sino Biological, Inc., Bejing,China; Cat #10428-H09H) and recombinant mouse Trop-2 with a His-Tag(Sino Biological, Inc., Cat #50922-MO8H) were plated onto Ni-NTA Hissorbstrips (Qiagen GmbH Cat #35023) at 1 μg for 1 h at room temperature. Theplate was washed four times with PBS-Tween (0.05%) wash buffer. Serialdilutions of hRS7 were made in 1% BSA-PBS dilution buffer to a testrange of 0.1 ng/mL to 10 μg/mL. The plates were then incubated for 2 hat room temperature before being washed four times followed by theaddition of a peroxidase conjugated secondary antibody (goat anti-human,Fc fragment specific; Jackson Immunoresearch Cat #109-036-098). After a45-min incubation, the plate was washed and a substrate solution(o-phenylenediamine dihydrochloride (OPD); Sigma, Cat #P828) added toall the wells. Plates were incubated in the dark for 15 min before thereaction was stopped with 4N sulfuric acid. The plates were read at 450nm on Biotek ELX808 plate reader. Data were analyzed and graphed usingPrism GraphPad Software (v4.03) (Advanced Graphics Software, Inc.;Encinitas, Calif.).

In Vitro Cell Binding—

LumiGLO Chemiluminescent Substrate System (KPL, Gaithersberg, Md.) wasused to detect antibody binding to cells. Briefly, cells were platedinto a 96 black-well, flat-clear-bottom plate overnight. Antibodies wereserially diluted 1:2 and added in triplicate, yielding a concentrationrange from 0.03 to 66.7 nM. After incubating for 1 h at 4° C., the mediawas removed and the cells washed with fresh cold media followed by theaddition of a 1:20,000 dilution of goat-antihuman horseradishperoxidase-conjugated secondary antibody (Jackson Immunoresearch, WestGrove, Pa.) for 1 h at 4° C. The plates were again washed before theaddition of the LumiGLO reagent. Plates were read for luminescence usingan Envision plate reader (Perkin Elmer, Boston Mass.). Data wereanalyzed by non-linear regression to determine the equilibriumdissociation constant (KD). Statistical comparisons of KD-values weremade with Prism GraphPad Software (v4.03) (Advanced Graphics Software,Inc.; Encinitas, Calif.) using an F-Test on the best-fit curves for thedata. Significance was set at P<0.05.

Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)—

A four-hour LDH-release assay was performed to evaluate ADCC activityelicited by IMMU-132, hRS7 IgG, hLL2-SN-38 and hLL2 IgG (hLL2 arenon-binding anti-CD22 conjugates for the solid tumor cell lines).Briefly, target cells (MDA-MB-468, NIH:OVCAR-3, or BxPC-3) were platedat 1×10⁴ cells/well in a 96-well, black, flat-bottom plate and incubatedovernight. The next day, peripheral blood mononuclear effector cells(PBMCs) were freshly isolated from a donor and added to assigned wellson the reaction plate at an E:T ratio of 50:1. Acquisition of humanPBMCs was done under the approval of the New England InstitutionalReview Board (Newton, Mass.). Test reagents were added to their assignedwells at a final concentration of 33.3 nM. One set of wells receivedADCC assay medium alone for background control and another set of wellsreceived cells alone plus TritonX100 for maximum cells lysis control.The plate was incubated for 4 h at 37° C. After 4 h, target cell lysiswas assessed by a homogenous fluorometric LDH release assay (CytoTox-One Homogenous Membrane Integrity Assay; Promega, Cat. G7891).

The plates were read (544 nm-590 nm) using an Envision plate reader(PerkinElmer LAS, Inc.; Shelton, Conn.). Data were analyzed by MicrosoftExcel. Percent specific lysis was calculated as follows:

${\%\mspace{14mu}{Specific}\mspace{14mu}{Lysis}} = {\frac{{Experimental} - \left( {{Effector} + {{Target}\mspace{14mu}{Control}}} \right)}{{{Max}.\mspace{11mu}{Lysis}} - \left( {{Target}\mspace{14mu}{Control}} \right)} \times 100}$where:

Experimental: effector+target cells+antibody

Effector+Target Control: effector+target cells

Max. Lysis: target cells+Triton-X100

Target Control: target cells only

Surface Plasmon Resonance Binding (BIACORE)—

Briefly, rhTrop-2/TACSTD2 (Sino Biological, Inc.) or recombinant humanneonatal receptor (FcRn), produced as described (Wang et al., 2011, DrugMetab Dispos 39:1469-77), was immobilized with an amine coupling kit (GEHealthcare; Cat. No. BR-1000-50) on a CM5 sensor chip (GE Healthcare;Cat. #BR-1000-12) following manufacturer's instructions for alow-density chip. Three separate sets of dilutions of hRS7 IgG andIMMU-132 were made in running buffer (400 nM, 200 nM, 100 nM, 50 nM and25 nM). Each set would make up a separate run on the BIACORE (BIACORE-X;Biacore Inc., Piscataway, N.J.) and data were analyzed usingBIAevaluation Software (Biacore Inc., v4.1). Analysis was performed witha 1:1 (Langmuir) Binding Model and Fit, using all five concentrationpoints for each sample run to determine the best fit (lowest χ² value).The KD value was calculated using the formula KD=kd1/ka1, where kd1 isthe dissociation rate-constant and ka1 is the association-rate constant.

Immunohistological Assessment of the Distribution of Trop-2 inFormalin-Fixed, Paraffin-Embedded Tissues—

Tumor xenografts were taken from mice, fixed in 10% buffered formalinand paraffin-embedded. After de-paraffination, 5 μm sections wereincubated with Tris/EDTA buffer (DaKo Target Retrieval Solution, pH 9.0;Dako, Denmark), at 95° C. for 30 min in a NxGen Decloaking Chamber(Biocare Medical, Concord, Calif.). Trop-2 was detected with a goatpolyclonal antihuman Trop-2 antibody at 10 μg/mL (R&D Systems,Minneapolis, Minn.) and stained with Vector VECTASTAINR ABC Kit (VectorLaboratories, Inc., Burlingame, Calif.). Normal goat antibody was usedas the negative control (R&D Systems, Minneapolis, Minn.). Tissues werecounterstained with hematoxylin for 6 seconds.

Trop-2 Surface Expression on Human Carcinoma Cell Lines—

Expression of Trop-2 on the cell surface is based on flow cytometry.Briefly, cells were harvested with Accutase Cell Detachment Solution(Becton Dickinson (BD), Franklin Lakes, N.J.; Cat. No. 561527) andassayed for Trop-2 expression using QuantiBRITE PE beads (BD Cat. No.340495) and a PE-conjugated anti-Trop-2 antibody (eBiosciences, Cat. No.12-6024) following the manufactures' instructions. Data were acquired ona FACSCalibur Flow Cytometer (BD) with CellQuest Pro software. Stainingwas analyzed with Flowjo software (Tree Star, Ashland Oreg.).

Pharmacokinetics—

Naive female NCr nude (nu/nu) mice, 8-10 weeks old, were purchased fromTaconic Farms (Germantown, N.Y.). Mice (N=5) were injected i.v. with 200μg of IMMU-132, parental hRS7, or modified hRS7-NEM (hRS7 treated withTCEP and conjugated with N-ethylmaleimide). Animals were bled viaretroorbital plexis at 30-min, 4-, 24-, 72- and 168-h post-injection.ELISA was utilized to determine serum concentrations of total hRS7 IgGby competing for the binding to an anti-hRS7 IgG idiotype antibody witha horseradish peroxidase conjugate of hRS7. Serum concentrations ofintact IMMU-132 were determined using an anti-SN-38 antibody to captureand a horseradish peroxidase-conjugated anti-hRS7 IgG antibody todetect. Pharmacokinetic (PK) parameters were computed bynoncompartmental analysis using Phoenix WinNonlin software (version 6.3;Pharsight Corp., Mountainview, Calif.).

Assessment of Double-Stranded DNA Breaks In Vitro—

For drug activity testing, BxPC-3 cells were seeded in 6-well plates at5×105 cells/well and held at 37° C. overnight. After 10 min cooling onice, cells were incubated with IMMU-132, hA20-SN-38, or hRS7-IgG at thefinal concentration of 20 μg/ml for 30 min on ice, washed three timeswith fresh media, and then returned to 37° C. to continue cultureovernight. The following morning, cells were trypsinized briefly, spundown, stained with Fixable Viability Stain 450 (BD Biosciences, SanJose, Calif.), washed with 1% BSA-PBS, and then fixed in 4% formalin for15 min, washed again and permeabilized in 0.15% Triton-X100 in PBS foranother 15 min. After washing twice with 1% BSA-PBS, cells wereincubated with mouse anti-γH2AX-AF488 (EMD Millipore Corporation,Temecula, Calif.) for 45 min at 4° C. The signal intensity of γH2AX wasmeasured by flow cytometry using a BD FACSCanto (BD Biosciences, SanJose, Calif.).

In Vivo Therapeutic Studies—

NCr female athymic nude (nu/nu) mice, 4-8 weeks old, were purchased fromTaconic Farms (Germantown, N.Y.). NCI-N87 gastric tumor xenografts wereestablished by harvesting cells from tissue culture and making a finalcell suspension 1:1 in matrigel (BD Bioscience; San Jose, Calif.), witheach mouse receiving a total of 1×10⁷ cells s.c. in the right flank. ForBxPC-3. xenografts of 1 g were harvested, and a tumor suspension made inHBSS to a concentration of 40% tumor w/v. This suspension was mixed 1:1with matrigel for a final tumor suspension of 20% w/v. Mice were theninjected with 300 μL s.c. Tumor volume (TV) was determined bymeasurements in two dimensions using calipers, with volumes defined as:L×w²/2, where L is the longest dimension of the tumor and w theshortest. For IHC, tumors were allowed to grow to approximately 0.5 cm³before the mice were euthanized and the tumors removed, formalin-fixedand paraffin-embedded. For therapy studies, mice were randomized intotreatment groups and therapy begun when tumor volumes were approximately0.25 cm³. Treatment regimens, dosages, and number of animals in eachexperiment are described in the Results and in the Figure legends. Thelyophilized IMMU-132 and control ADC (hA20-SN-38) were reconstituted anddiluted as required in sterile saline.

Mice were euthanized and deemed to have succumbed to disease once tumorsgrew to greater than 1.0 cm³ in size. Best responses to therapy weredefined as: partial response, shrinking >30% from starting size; stabledisease, tumor volumes shrinking up to 29% or increase no greater than20% of initial size; progression, tumors increase >20% either from theirstarting size or from their nadir. Time to progression (TTP) wasdetermined as time post-therapy initiation when the tumor grew more than20% in size from its nadir.

Statistical analysis of tumor growth was based on area under the curve(AUC). Profiles of individual tumor growth were obtained throughlinear-curve modeling. An f-test was employed to determine equality ofvariance between groups prior to statistical analysis of growth curves.A two-tailed t-test was used to assess statistical significance betweenthe various treatment groups and controls, except for the salinecontrol, where a one-tailed t-test was used (significance at P<0.05).Survival studies were analyzed using Kaplan-Meier plots (log-rankanalysis), using the Prism GraphPad Software (v4.03) software package(Advanced Graphics Software, Inc., Encinitas, Calif.).

Immunoblotting—

Cells (2×10⁶) were plated in 6-well plates overnight. The following daythey were treated with either free SN-38 (dissolved in DMSO) or IMMU-132at an SN-38 concentration equivalent to 0.4 μg/mL (1 μM). Parental hRS7was used as a control for the ADC. Cells were lysed in buffer containing10 mM Tris, pH 7.4, 150 mM NaCl, protease inhibitors and phosphataseinhibitors (2 mM Na2PO4, 10 mM NaF). A total of 20 μg protein wasresolved in a 4-20% SDS polyacrylamide gel, transferred onto anitrocellulose membrane and blocked by 5% non-fat milk in 1×TBS-T(Tris-buffered saline, 0.1% Tween-20) for 1 h at room temperature.Membranes were probed overnight at 4° C. with primary antibodiesfollowed by 1-h incubation with antirabbit secondary antibody (1:2500)at room temperature. Signal detection was done using a chemiluminescencekit (Supersignal West Dura, Thermo Scientific; Rockford, Ill.) with themembranes visualized on a Kodak Image Station 40000R. Primary antibodiesp21Waf1/Cip1 (Cat. No. 2947), Caspase-3 (Cat. No. 9665), Caspase-7 (Cat.No. 9492), Caspase-9 (Cat. No. 9502), PARP (Cat. No. 9542), (3-actin(Cat. No. 4967), pJNK1/2 (Cat. No. 4668), JNK (Cat. No. 9258), and goatanti-rabbit-HRP secondary antibody (Cat. No. 7074) were obtained fromCell Signal Technology (Danvers, Mass.).

Results

Trop-2 Expression Levels in Multiple Solid Tumor Cell Lines—

Surface expression of Trop-2 is evident in a variety of human solidtumor lines, including gastric, pancreatic, breast, colon, and lung(Table 12). There is no one tumor type that had higher expression aboveany other, with variability observed within a given tumor cell type. Forexample, within gastric adenocarcinomas, Trop-2 levels ranged from verylow 494±19 (Hs 746T) to high 246,857±64,651 (NCI-N87) surface moleculesper cell.

Gastrointestinal tumor xenografts stained for Trop-2 expression showedboth cytoplasmic and membrane staining (not shown). Staining intensitycorrelated well with the results for surface Trop-2 expressiondetermined by FACS analysis. For the pancreatic adenocarcinomas, allthree had homogenous staining, with BxPC-3 representing 2+ to 3+staining. NCI-N87 gastric adenocarcinoma had a more heterogeneousstaining pattern, with 3+ staining of the apical lining of the glandsand less pronounced staining of surrounding tumor cells. COLO 205demonstrated only very focal 1+ to 2+ staining, whereas HT-29 showedvery rare 1+ staining of a few cells.

TABLE 12 Trop-2 surface expression levels in various solid tumor linesvia FACS analysis. a Number of Surface Trop-2 Molecules per Cell CellLine Mean ± SD Gastric NCI-N87 246,857 ± 64,651 AGS  53,756 ± 23,527 Hs746T 494 ± 19 Pancreatic BxPC-3 493,773 ± 97,779 CFPAC-1 162,871 ±28,161 Capan-1 157,376 ± 36,976 HPAF-II 115,533 ± 28,627 Breast (TN)MDA-MB-468 301,603 ± 29,470 HCC38 181,488 ± 69,351 HCC1806  91,403 ±20,817 MDA-MB-231 32,380 ± 5,460 Breast SK-BR-3 (HER2+) 328,281 ± 47,996MCF-7 (ER2+) 110,646 ± 17,233 Colon COLO 205 58,179 ± 6,909 HT-29  68 ±17 NSCLC Calu-3 128,201 ± 50,708 Sq. Cell Lung SK-MES-1 29,488 ± 5,824Acute T-Cell Leukemia Jurkat 0 a Three separate assays were performed,with the mean and standard deviation provided.

IMMU-132 Binding Characteristics—

To further demonstrate that hRS7 does not cross-react with murineTrop-2, an ELISA was performed on plates coated with either recombinantmurine Trop-2 or human Trop-2 (not shown). Humanized RS7 specificallybound only to the human Trop-2 (KD=0.3 nM); there was nocross-reactivity with the murine Trop-2. Control polyclonal rabbitanti-murine Trop-2 and antihuman Trop-2 antibodies did cross-react andbound to both forms of Trop-2 (data not shown).

IMMU-132 binding to multiple cell lines was examined, with comparison toparental hRS7 as well as to modified hRS7, hRS7-NEM (hRS7 treated withTCEP and conjugated with N-ethylmaleimide) (not shown). In all cases,calculated KD-values were in the sub-nanomolar range, with nosignificant differences between hRS7, IMMU-132, and hRS7-NEM within agiven cell line.

Comparisons in binding of IMMU-132 and hRS7 were further investigatedusing surface plasmon resonance (BIACORE) analysis (not sown). Alow-density Trop-2 biosensor chip (density=1110 RU) was utilized withrecombinant human Trop-2. Not only did three independent binding runsdemonstrate that IMMU-132 is not affected adversely by theSN-38-conjugation process, but it demonstrated a higher binding affinityto Trop-2 than hRS7 (0.26±0.14 nM vs. 0.51±0.04 nM, respectively;P=0.0398).

Mechanism of Action: ADCC and Intrinsic Apoptosis Signaling Pathways—

ADCC activity of IMMU-132 was compared to hRS7 in three different celllines, TNBC (MDAMB-468), ovarian (NIH:OVCAR-3), and pancreatic (BxPC-3)(FIG. 21). In all three, hRS7 significantly mediated cell lysis comparedto all other treatments, including IMMU-132 (P<0.0054). ADCC decreasedby more than 60% when IMMU-132 was used to target the cells as comparedto hRS7. For example, in MDA-MB-468, specific lysis mediated by hRS7 was29.8±2.6% versus 8.6±2.6% for IMMU-132 (FIG. 21A; P<0.0001). Similarloss in ADCC activity was likewise observed in NIH:OVCAR-3 and BxPC-3(FIGS. 21B and 21C; P<0.0001 and P<0.0054; respectively). Thisdiminished ADCC activity appears to be the result of changes to theantibody during the conjugation process, since this same loss inspecific cell lysis was evident with hRS7-NEM, which lacks theCL2A-SN-38 linker, having the cysteines blocked instead withN-ethylmaleimide (FIG. 21C). There is no CDC activity associated withhRS7 or IMMU-132 (data not shown).

IMMU-132 has been shown previously to mediate the up-regulation of earlypro-apoptosis signaling events (p53 and p21WAF1/Cip1), ultimatelyleading to the cleavage of PARP20. In order to better define theapoptotic pathway utilized by IMMU-132, the NCI-N87 human gastriccarcinoma and BxPC-3 pancreatic adenocarcinoma cell lines were exposedto 1 μM of free SN-38 or the equivalent amount of IMMU-132 (not shown).Both free SN-38 and IMMU-132 mediate the up-regulation of p21WAF1/Cip1,though it is not until 48 h that the up-regulation between the NCI-N87cells exposed to free SN-38 versus IMMU-132 are the same (not shown),whereas in BxPC-3 maximum up-regulation is evident within 24 h (notshown). Both free SN-38 and IMMU-132 demonstrate cleavage ofpro-caspase-9 and -7 within 48 h of exposure. Procaspase-3 is cleaved inboth cell lines with the highest degree of cleavage observed after 48 h.Finally, both free SN-38 and IMMU-132 mediated PARP cleavage. This firstbecomes evident at 24 h, with increased cleavage at 48 h. Takentogether, these data confirm that the SN-38 contained in IMMU-132 hasthe same activity as free SN-38.

In addition to these later apoptosis signaling events, an earlier eventassociated with this pathway, namely the phosphorylation of JNK (pJNK),is also evident in BxPC-3 cells exposed for a short time to either freeSN-38 or IMMU-132, but not naked hRS7 (not shown). Increased amounts ofpJNK are evident by 4 h, with no appreciable change at 6 h. There is ahigher intensity of phosphorylation in the cells exposed to free-SN-38as compared to IMMU-132, but both are substantially higher thancontrols. As an end-point for the mechanism of action of IMMU-132,measurements of dsDNA breaks were made in BxPC-3 cells. Exposure ofBxPC-3 to IMMU-132 for only 30 min resulted in a greater than two-foldinduction of γH2AX when compared to a non-targeting control ADC (Table13). Approximately 70% of the cells were positive for γH2AX stainingversus <20% for naked hRS7, hA20-SN-38 irrelevant ADC, and untreatedcontrols (P<0.0002).

TABLE 13 IMMU-132-mediated dsDNA breaks in BxPC-3: γH2AX induction.^(a)Treatment Mean Fluorescence Intensity Percent Positive Untreated 2516 ±191 18.8 ± 6.3 hRS7 2297 ± 18  13.0 ± 0.6 hA20-SN-38 2246 ± 58  12.7 ±2.4 IMMU-132 5349 ± 234 69.0 ± 4.1 ^(a)IMMU-132 vs. all 3 controltreatments, P < 0.0002 (onetailed t-Test; N = 3).

Pharmacokinetics of IMMU-132—

Binding to the human neonatal receptor (FcRn) was determined by BIACOREanalysis (not shown). Using a low-density FcRn biosensor chip(density=1302 RU), three independent binding runs at five differentconcentrations (400 to 25 nM) were conducted for each agent. Overall,both hRS7 and IMMU-132 demonstrate KD-values in the nanomolar range(92.4±5.7 nM and 191.9±47.6 nM, respectively), with no significantdifference between the two. Mice were injected with IMMU-132, with theclearance of IMMU-132 versus the hRS7 IgG compared to the parental hRS7using two ELISAs (FIG. 22). Mice injected with hRS7 demonstrated abiphasic clearance pattern (FIG. 22A) that was similar to what wasobserved for the hRS7 targeting portion of IMMU-132 (FIG. 22B), withalpha and beta half-lives of approximately 3 and 200 h, respectively. Incontrast, a rapid clearance of intact IMMU-132 was observed with ahalf-life of 11 h and mean residence time (MRT) of 15.4 h (FIG. 22C).

To further confirm that disruption of interchain disulfide bonds doesnot alter the PK of the targeting antibody, the PK of parental hRS7 wascompared to modified hRS7 (hRS7-NEM). There were no significantdifferences noted between either agent in terms of half-life, Cmax, AUC,clearance, or MRT (not shown).

IMMU-132 Efficacy in Human Gastric Carcinoma Xenografts—

Efficacy of IMMU-132 has been demonstrated previously in non-small-celllung, colon, TNBC, and pancreatic carcinoma xenograft models (Cardilloet al., 2011, Clin Cancer Res 17:3157-69; Goldenberg et al., Posterpresented at San Antonio Breast Cancer Symposium, December 9-13, AbstrP5-19-08). To further extend these findings to other gastrointestinalcancers, IMMU-132 was tested in mice bearing a human gastric carcinomaxenograft, NCI-N87 (FIG. 23). Treatment with IMMU-132 achievedsignificant tumor regressions compared to saline and non-targeting hA20(anti-CD20)-SN-38 ADC controls (FIG. 23A; P<0.001). There were 6 of 7mice in the IMMU-132 group that were partial responders that lasted formore than 18 days after the last therapy dose was administered to theanimals. This resulted in a mean time to progression (TTP) of 41.7±4.2days compared to no responders in the control ADC group, with a TTP of4.1±2.0 days (P<0.0001). Overall, the median survival time (MST) forIMMU-132-treated mice was 66 days versus 24 days for control ADC and 14days for saline control animals (FIG. 23B; P<0.0001).

Clinically-Relevant Dosing Schemes—

The highest repeated doses tolerated of IMMU-132 currently being testedclinically are 8 and 10 mg/kg given on days 1 and 8 of 21-day cycles. Ahuman dose of 8 mg/kg translates to a murine dose of 98.4 mg/kg, orapproximately 2 mg to a 20 g mouse. Three different dose schedules offractionated 2 mg of IMMU-132 were examined in a human pancreaticadenocarcinoma xenograft model (BxPC-3). This total dose wasfractionated using one of three different dosing schedules: one groupreceived two IMMU-132 doses of 1 mg (therapy days 1 and 15), onereceived four doses of 0.5 mg (therapy days 1, 8, 22, and 29), and thefinal group eight doses of 0.25 mg (therapy days 1, 4, 8, 11, 22, 25,29, and 32). All three dosing schemes provided a significant anti-tumoreffect when compared to untreated control animals, both in terms oftumor growth inhibition and overall survival (FIG. 24A; P<0.0009 andP<0.0001, respectively). There are no significant differences in TTPbetween the three different treatment groups, which ranged from22.4±10.1 days for the 1-mg dosing group to 31.7±14.5 days for the0.25-mg dosing group (TTP for untreated control group=5.0±2.3 days).

A similar dose schedule experiment was performed in mice bearing NCI-N87human gastric tumor xenografts (FIG. 24B). All three dose schedules hada significant anti-tumor effect when compared to untreated control micebut were no different from each other (AUC; P<0.0001). Likewise, interms of overall survival, while all three dose schedules provided asignificant survival benefit when compared to untreated control(P<0.0001), there were no differences between any of these threedifferent schedules.

To further discriminate possible dosing schemes, mice bearing NCI-N87tumors were subjected to chronic IMMU-132 dosing in which mice received0.5 mg injections of IMMU-132 once a week for two weeks followed by oneweek off before starting another cycle (FIG. 24C), as in the currentclinical trial schedule. In all, four treatment cycles were administeredto the animals.

This dosing schedule slowed tumor growth with a TTP of 15.7±11.1 daysversus 4.7±2.2 days for control ADC-treated mice (P=0.0122). Overall,chronic dosing increased the median 19 survival 3-fold from 21 days forcontrol ADC-treated mice to 63 days for those animals administeredIMMU-132 (P=0.0001). Importantly, in all these different dosing schemeevaluations, no treatment-related toxicities were observed in the miceas demonstrated by no significant loss in body weight (data not shown).

DISCUSSION

In a current Phase I/II clinical trial (ClinicalTrials.gov,NCT01631552), IMMU-132 (sacituzumab govitecan) is demonstratingobjective responses in patients presenting with a wide range of solidtumors (Starodub et al., 2015, Clin Cancer Res 21:3870-78). As thisPhase I/II clinical trial continues, efficacy of IMMU-132 needs to befurther explored in an expanding list of Trop-2-positive cancers.Additionally, the uniqueness of IMMU-132, in contrast to otherclinically relevant ADCs that make use of ultratoxic drugs, needs to befurther elucidated as we move forward in its clinical development.

The work presented here further characterizes IMMU-132 and demonstratesits efficacy against gastric and pancreatic adenocarcinoma atclinically-relevant dosing schemes. The prevailing view of a successfulADC is that it should use an antibody recognizing an antigen with hightumor expression levels relative to normal tissue and one thatpreferably internalizes when bound to the tumor cells (Panowski et al.,2014, mAbs 6:34-45). All of the currently approved ADCs have used anultra-toxic drug (pM IC50) coupled to the antibody by a highly stablelinker at low substitution ratios (2-4 drugs per antibody). IMMU-132diverges from this paradigm in three main aspects: (i) SN-38, amoderately cytotoxic drug (nM IC50), is used as the chemotherapeuticagent, (ii) SN-38 is conjugated site-specifically to 8 interchain thiolsof the antibody, yielding a substitution of 7.6 drugs per antibody, and(iii) a carbonate linker is used that is cleavable at low pH, but willalso release the drug with a half-life in serum of ˜24 h (Cardillo etal., 2011, Clin Cancer Res 17:3157-69). IMMU-132 is composed of anantibody that internalizes after binding to an epitope, as we haveshown, that is specific for human Trop-2, which is highly expressed onmany different types of epithelial tumors, as well as at lowerconcentrations in their corresponding normal tissues (Shih et al., 1995,Cancer Res 55:5857s-63s). Despite the presence in normal tissues, priorstudies in monkeys, which also express Trop-2 in similar tissues,indicated relatively mild and reversible histopathological changes evenat very high doses where dose-limiting neutropenia and diarrheaoccurred, suggesting the antigen in the normal tissues was sequesteredin some manner, or that the use of a less toxic drug spared these normaltissues from severe damage (Cardillo et al., 2011, Clin Cancer Res17:3157-69).

Herein, we expanded an assessment of Trop-2 expression on multiple humansolid tumor lines, examining in vitro expression in a more quantitativemanner than reported previously, but also, importantly, in xenograftsthat illustrate Trop-2 expression ranging from homogenous (e.g., NCIN87)to very focal (e.g., COLO 205). Overall, surface expression levels ofTrop-2 determined in vitro correlated with staining intensity upon IHCanalysis of xenografts. It is particularly interesting that even in atumor like COLO 205, where there are only focal pockets ofTrop-2-expressing cells revealed by immunohistology, IMMU-132 was stillcapable of eliciting specific tumor regressions, suggesting that abystander effect may occur as a result of the release of SN-38 from theconjugate bound to the antigen-presenting cells (Cardillo et al., 2011,Clin Cancer Res 17:3157-69). Indeed, SN-38 readily penetrates cellmembranes, and therefore its local release within the tumormicroenvironment provides another mechanism for its entry into cellswithout requiring internalization of the intact conjugate. Importantly,the SN-38 bound to the conjugate remains in a fully active state;namely, it is not glucuronidated and would be in the lactone ring format the time of release (Sharkey et al., 2015, Clin Cancer Res,21:5131-8). This property is unique, distinguishing IMMU-132's abilityto localize a fully active form of SN-38 in a more selective manner thanany of the other slow-release SN-38 or irinotecan agents studied todate.

The Phase I clinical trial with IMMU-132 identified 8 to 10 mg/kg givenweekly for two weeks on a 21-day cycle for further investigation inPhase II (Starodub et al., 2015, Clin Cancer Res 21:3870-78). Patientswith a wide range of metastatic solid tumors, including pancreatic andgastric cancers, have shown extended periods of disease stabilizationafter relapsing to multiple prior therapies (Starodub et al., 2015, ClinCancer Res 21:3870-78; Starodub et al., 2014, J Clin Oncol 32:5s (SupplAbstr 3032)). Additional studies in xenograft models were undertaken todetermine if different dosing schedules may be more efficacious. To thisend, the equivalent to the human dose of 8 mg/kg (mouse dose of 98.4mg/kg) was fractionated over three different dosing schedules, includingevery other week, weekly, or twice-weekly on a 21-day cycle. In thepancreatic and gastric tumor models, no significant difference intherapeutic responses were observed for all three schedules, with tumorsprogressing only after therapy was discontinued. Therefore, these datasupport the continued use of the once-weekly dosing regimen currentlybeing pursued clinically.

With clinical trials recommending an IMMU-132 each treatment dose of 8to 10 mg/kg (Starodub et al., 2015, Clin Cancer Res 21:3870-78), it wasimportant to examine whether the antibody alone might contribute to theIMMU-132's activity. Previous studies in nude mice-human xenograftmodels had included unconjugated hRS7 IgG alone (e.g., repeated doses of25 to 50 mg/kg), with no evidence of therapeutic activity (Cardillo etal., 2011, Clin Cancer Res 17:3157-69); however, studies in mice cannotalways predict immunological functionality. ADCC activity of hRS7 invitro has been reported in Trop-2-positive ovarian and uterinecarcinomas (Raji et al., 2011, J Exp Clin Cancer Res 30:106; Bignotti etal., 2011, Int J Gynecol Cnacer 21:1613-21; Varughese et al., 2011, Am JObstet Gynecol 205:567; Varughese et al., 2011, Cancer 117:3163-72). Weconfirmed unconjugated hRS7 ADCC activity in three different cellslines, but found IMMU-132 lost 60-70% of its effector function. Sincethe reduced/NEM-blocked IgG has a similar loss of ADCC activity, itappears that the attachment of the CL2A-SN-38 component was not, initself, responsible.

Antibodies also can elicit cell death by acting on various apoptoticsignaling pathways. However, we did not observe any effects of theunconjugated antibody in a number of apoptotic signaling pathways, butinstead noted IMMU-132 elicited similar intrinsic apoptotic events asSN-38. Early events include the phosphorylation of JNK1/2 as well as theup-regulation of p21WAF1/Cip1 leading to the activation of caspase-9,-7, and -3, with the end result of PARP cleavage and significant levelsof dsDNA breaks, as measured by increased amounts of phosphorylatedhistone H2AX (γH2AX)₄₁. These data suggest that IMMU-132's primarymechanism of action is related to SN-38.

Surface plasmon resonance (BIACORE) analysis did not detect asignificant difference in IMMU-132's binding to the human neonatalreceptor (FcRn), despite the average binding levels being ˜2-fold lowerfor IMMU-132. FcRn binding has been linked to an extended IgG half-lifein serum (Junghans & Anderson, 1996, Proc Natl Acad Sci USA 93:5512-16),but because an antibody's affinity for FcRn in vitro may not correlatewith in vivo clearance rates (Datta-Mannan et al., 2007, J Biol Chem282:1709-17), the overall importance of this finding is unknown.Previous experiments in tumor-bearing mice using ¹¹¹In-DTPA-IMMU-132revealed that the conjugate cleared at a somewhat faster rate from theserum than ¹¹¹In-DTPA-hRS7, although both had similar tumor uptake(Cardillo et al., 2011, Clin Cancer Res 17:3157-69). In the currentstudies, an ELISA assay that also measured the clearance of the IgGcomponent found IMMU-132 and the reduced and NEM-blocked IgG cleared atsimilar rates as unconjugated hRS7, suggesting that the coupling to theinterchain disulfides does not destabilize the antibody. As expected,when using an ELISA that monitored the clearance of the intact conjugate(capturing using an anti-SN-38 antibody and probe with an anti-idiotypeantibody), its clearance rate was faster than when monitoring only theIgG component. This difference simply reflects SN-38's release from theconjugate with a half-life of ˜1 day. We also have examined theclearance rates of hRS7-SN-38 conjugates prepared at differentsubstitution levels by ELISA, and again found no appreciate differencein their clearance rates (Goldenberg et al., 2015, Oncotarget8:22496-512). Overall, these data suggest that mild reduction of theantibody, with the subsequent site-specific modification of some or allinterchain disulfides, has minimal if any impact on the serum clearanceof the IgG, but IMMU-132's overall clearance rate will be definedlargely by the rate of release of SN-38 from the linker.

Additionally, extensive cell-binding experiments demonstrated nosignificant difference in the binding of IMMU-132, the unconjugatedantibody, or the NEM-modified antibody, suggesting that thesite-specific linkage to the interchain disulfides protects theantigen-binding properties of the antibody. Interestingly, when analyzedby BIACORE, which more accurately measures the on-rate and off-rate inaddition to overall affinity, IMMU-132 had a significant 2-foldimprovement in calculated KD-values for Trop-2 binding when compared tonaked hRS7.

We speculate that this improvement may be result of the addedhydrophobicity when SN-38 is conjugated to the antibody. Hydrophobicresidues, as well as hydrophobicity of enclosed regions of proteinbinding sites, have been shown to impart a stronger affinity for theepitope (Park et al., 2000, Nat Biotechnol 18:194-98; Berezov et al.,2001, J Med Chem 44:2565-74; Young et al., 2007, Proc Natl Acad Sci USA104:808013). These regions do not have to be at the protein-proteininterface, but can lie in surrounding, less energetically contactresidues (Li et al., 2005, Structure 13:297-307). While none of theSN-38 conjugation sites are present in the complement-determiningregions (CDR) of hRS7, the prospects that the SN-38 on the antibody maydisplace some of the water molecules around the epitope, resulting inthe improved binding affinity observed for IMMU-132 relative to nakedhRS7, cannot be discounted.

Most efforts in ADC development have been directed towards using astable linker and an ultratoxic drug, with preclinical studiesindicating the specific optimal requirements for those conjugates(Panowski et al., 2014, mAbs 6:34-45; Phillips et al., 2008, Cancer Res68:9280-90). For example, a comparison of T-DM1 to another less stablederivative, T-SSPDM1, revealed that intact T-SSP-DM1 cleared at anapproximately 2-fold faster rate than T-DM-1 in non-tumor-bearing mice(Phillips et al., 2008, Cancer Res 68:9280-90; Erickson et al., 2012,Mol Cancer Ther 11:1133-42), with 1.5-fold higher levels of T-DM1compared to T-SSPDM1 in the tumors. Unexpectedly, and most interesting,was the finding that the amount of free, active maytansinoid catabolitesin the targeted tumors was very similar between the two ADCs (Ericksonet al., 2012, Mol Cancer Ther 11:1133-42).

In other words, T-SSP-DM1 was able to overcome its deficiencies inlinker stability due to the fact that the lower stability resulted inmore efficient release of the drug at the tumor than the more stableT-DM1. Not surprisingly, this equivalency of active drug-catabolitebetween the two ADCs in the tumors resulted in similar anti-tumoreffects in tumor-bearing animals. Ultimately, T-DM1 was chosen based ontoxicity issues that arise when using an ultra-toxic drug and lessstable linkers (Phillips et al., 2008, Cancer Res 68:9280-90). SinceSN-38 is at least a log-fold less toxic than these maytansines, itsrelease from the ADC is expected to have less toxicity. However, evenwith its release in serum, the amount of SN-38 localized in humangastric or pancreatic tumor xenografts was up to 136-fold higher than intumor-bearing mice injected with irinotecan doses that had >20-foldhigher SN-38 equivalents (Sharkey et al., 2015, Clin Cancer Res,21:5131-8). While we have tested more stable linkers in the developmentof IMMU-132, they were significantly less effective in xenograft tumormodels than IMMU-132 (Govindan et al., 2013, Mol Cancer Ther 12:968-78).

Similarly, linkers that released SN-38 more quickly (e.g., serumhalf-life of −10 h) also were less effective in xenograft models (Moonet al., 2008, J Med Chem 51:6916-26; Govindan et al., 2009, Clin ChemRes 15:6052-61), suggesting that there is an optimal window at which therelease of SN-38 leads to improved efficacy. Thus, current datademonstrate that IMMU-132 is a more efficient way to target and releasethe drug at the tumor than irinotecan.

Early clinical studies have shown encouraging objective responses invarious solid tumors, and importantly have indicated a better safetyprofile, with a lower incidence of diarrhea, than irinotecan therapy(Starodub et al., 2015, Clin Cancer Res 21:3870-78).

In summary, IMMU-132 (sacituzumab govitecan) is a paradigm-shift in ADCdevelopment. It uses a moderately-stable linker to conjugate 7-8molecules of the more tolerable active metabolite of irinotecan, SN-38,to an anti-Trop-2 antibody. Despite these seemingly counterintuitivecharacteristics vis-a-vis ultra-toxic ADCs, non-clinical studies havedemonstrated that IMMU-132 very effectively targets Trop-2-expressingtumors with significant efficacy and no appreciable toxicity. In earlyphase I/II clinical trials against a wide range of solid tumors,including pancreatic, gastric, TNBC, small-cell and non-small-cell lungcarcinomas, IMMU-132 is likewise exhibiting anti-tumor effects withmanageable toxicities in these patients, with no immune responses toeither the IgG or SN-38 detected, even after many months of dosing(Starodub et al., 2015, Clin Cancer Res 21:3870-78). Given the elevatedexpression of Trop-2 on such a wide variety of solid tumors, IMMU-132continues to be studied clinically, especially in advanced cancers thathave been refractory to most current therapy strategies.

Example 17. Further Results from Phase I/II Clinical Studies of IMMU-132

Triple-Negative Breast Cancer (TNBC)

The phase I/II clinical trial (NCT01631552) discussed in the Examplesabove has continued, accruing 56 TNBC patients who were treated with 10mg/kg. The patient population had previously been extensively treatedbefore initiating IMMU-132 therapy, with at least 2 prior lines oftherapy including taxane treatment. Previous treatments includedcyclophosphamide, doxorubicin, carboplatin, gemcitabine, capecitabine,eribulin, cisplatinum, anastrozole, vinorelbine, bevacizumab andtamoxifen. Despite this extensive treatment history TNBC patientsresponded well to IMMU-132, with 2 confirmed complete responses (CR), 13partial responses (PR) and 25 stable disease (SD), for an objectiveresponse rate of 29% (15/52) (FIG. 25). Adding the incidence of CR plusPR plus SD, treatment in TNBC resulted in a a 71% favorable responserate for IMMU-132 treated patients (FIG. 26). The median time toprogression in this heavily pretreated population of TNBC patients was9.4 months, with a range of 2.9 to 14.2 months to date. However, 72% ofpatients in the study were still ongoing treatment. The progression-freesurvival in this group of patients is shown in FIG. 27.

Metastatic NSCLC

The clinical trial is also ongoing for patients with metastaticnon-small cell lung cancer (NSCLC), with 29 assessable patients accruedto date, who were treated with 8 or 10 mg/kg IMMU-132. The bestresponses by RESIST 1.1 criteria are shown in FIG. 28. Out of 29patients, there were 8 PR and 13 SD. The time to progression for NSCLCpatients is shown in FIG. 29, which shows that 21/33 (64%) of NSCLCpatients exhibited PR or SD. The median time to progression was 9/4months, with a range from 1.8 to 15.5+ months and 47% of patients stillundergoing treatment. Progression-free survival in NSCLC patientstreated with 8 or 10 mg/kg IMMU-132 is shown in FIG. 30. Median PFS was3.4 months at 8 mg/kg and 3.8 months at 10 mg/kg. However, studies arestill ongoing and the median progression-free survival numbers arelikely to improve.

Metastatic SCLC

Comparable results in metastatic SCLC patients are shown in FIG. 31-33.Best response by RECIST 1.1 for metastatic SCLC patients treated with 8or 10 mg/kg IMMU-132 showed 6 PR and 8 SD out of 25 assessable patients(FIG. 31). Time to progression (FIG. 32) showed a median of 4.9 months,with a range of 1.8 to 15.7+ months and 7 patients still undergoingtreatment with IMMU-132. The progression free survival (FIG. 33) showeda median PFS of 2.0 months at 8 mg/kg and 3.6 months at 10 mg/kg. Themedian OS was 8.1 months at 8 mg/kg and could not be determined yet for10 mg/kg.

Urothelial Cancer

Similar results were obtained with urothelial cancer patients treatedwith 8 or 10 mg/kg IMMU-132. The best response daeta for 11 assessablepatients showed 6 PR and 2 SD (FIG. 34). Time to progression (FIG. 35)showed a median of 8.1 months, with a range of 3.6 yo 9.7+ months.

In summary, the continuing phase I/II clinical trial shows superiorefficacy of IMMU-132, when administered at the recited dosages of ADC,in at least TNBC, NSCLC, SCLC and urothelial cancers. The superiortherapeutic effect in these heavily pretreated and resistant metastaticcancers occurred without inducing severe toxicities that might precludeclinical use. IMMU-132 showed an acceptable safety profile in heavilypretreated patients with diverse solid cancers, and a median of 2-5prior therapies. Only neutropenia showed an incidence of greater than20% of the patient population for Grade 3 or higher adverse reactions.The study further demonstrates that repeated doses of IMMU-132 may beadministered to human patients, at therapeutic dosages, without evokinginterfering host anti-IMMU-132 antibodies. These results demonstrate thesafety and utility of IMMU-132 for treating diverse Trop-2 positivecancers in human patients.

Example 18. Combination Therapy with Anti-Trop-2 ADC and ABCG2 InhibitorSummary

As discussed above, IMMU-132 is showing promising therapeutic results ina phase II trials of patients with metastatic triple-negative breast andother cancers who were heavily pretreated (ClinicalTrials.gov,NCT01631552), and who express high levels of Trop-2. This novelTrop-2-targeting humanized antibody is conjugated with 7.6 moles ofSN-38, the active form of irinotecan, by a proprietary linker, and isless glucuronidated in vivo than irinotecan, accounting forsignificantly lower incidence of diarrhea in patients treated with theagent. Since members of the ATP-binding cassette (ABC) transporters,notably P-gp (ABCB1) and BCRP (ABCG2), confer resistance by active drugefflux, which is a frequent cause of treatment failure, we examined theuse of known inhibitors of ABC transporters for improving therapeuticresults of IMMU-132 by overcoming SN-38 resistance in preclinicalstudies.

Two SN-38-resistant human cancer cell lines, MDA-MB-231-S120 (breastcancer) and NCI-N87-S120 gastric cancer), were generated by continuousexposure in culture media with stepwise increased concentrations ofSN-38 (up to 120 nM) and analyzed by flow cytometry for functionalactivities of ABCG2 and ABCB1, immunoblotting and RT-qPCR for theexpression of ABCG2 at both protein and mRNA levels, and MTS assays forthe potency of SN-38 alone or in combination with a modulator of ABCtransporters. Also, a xenograft model of NCI-N87-S120 in SCID mice wasused for assessing antitumor activity in vivo.

MDA-MB-231-S120 and NCI-N87-S120 displayed reduced sensitivity to SN-38,with IC₅₀ values approximately 50-fold higher than parental MDA-MB-231and NCI-N87 cells. The increase in drug resistance of both S-120 cellsis associated with the expression of functional ABCG2, but not ABCB1; a2- to 4-fold higher topoisomerase-1; and a lack of continuingaccumulation of double-strand breaks as observed for the parental cellsover a period of 3 h by monitoring the levels of phosphorylated H2AX(γH2AX). Importantly, treatment of both S-120 cells with known ABCG2inhibitors (fumitremorgin C, Ko143, GF120918, and YHO-13351) restoredtoxicity of SN-38 in vitro, and the combination of YHO-13351 withIMMU-132 improved the median survival of mice bearing NCI-N87-S120xenografts compared to IMMU-132 alone. We conclude that combinationtherapy of IMMU-132 and YHO-13351, or other inhibitors of ABCtransporters, is of use for treatment of cancers that are resistant tocamptothecin chemotherapeutic agents and/or camptothecin-conjugatedADCs.

Materials and Methods

Cell Lines and Cultures.

Human cancer cell lines (MDA-MB-231, breast; NCI-N87, stomach; A549,lung; HCT15, colon) were purchased from the American Type CultureCollection (ATCC) with authentication by Short Tandem Repeat profiling.Each cell line was maintained according to the recommendations of ATCCand routinely tested for mycoplasma using MYCOALERT™ mycoplasmadetection kits (Lonza). The two SN-38-resistant cell lines,MDA-MB-231-5120 and NCI-N87-S120, were established by continuousexposure of the parental cells to stepwise increased concentrations ofSN-38 from 6 pM to 120 nM over a period of approximately 2 years. Inaddition, a revertant cell line (NCI-N87-S120-REV) was obtained afterculturing NCI-N87-S120 in SN-38-free medium for 4 months. The S-120clones were maintained in medium containing 120 nM of SN-38, butcultured in drug-free medium for 7 to 14 days prior to any experiment.All cells were grown at 37° C. as monolayer cultures in a humidifiedatmosphere of 5% CO₂.

Antibodies, IMMU-132, and reagents. Polyclonal rabbit anti-ABCG2antibody (#4477) was purchased from Cell Signaling and murineanti-γH2AX-AF488 (05-636-AF488) from EMD Millipore. Pheophorbide A(PhA), fumitremorgin C (FTC), Ko143, YHO-13351, doxorubicin, paclitaxel,rhodamine 123, and verapamil were obtained from Sigma. SN-38, purchasedfrom Biddle Sawyer Pharma, was diluted to 1 mM in dimethyl sulfoxide(DMSO) and stored in aliquots at −20° C. Irinotecan-HCl injection waspurchased from AREVA Pharmaceuticals. The properties and preparation ofIMMU-132 have been described previously (Cardillo et al., 2011, ClinCancer Res 17:3157-69; Cardillo et al., 2015, Bioconjugate Chem26:919-31; Goldenberg et al., 2015, Oncotarget 6:22496-512; U.S. Pat.No. 7,999,083).

Functional assays of ABC transporters. Functional assays of ABCG2 withPhA as the substrate and FTC as the inhibitor were performed essentiallyas described by Robey et al (Robey et al., 2004, Cancer Res 64:1242-6).Briefly, trypsinized cells were incubated with 1 PhA or with acombination of 1 μM PhA and 10 μM FTC for 30 min at 37° C. in 5% CO₂,and washed. Subsequent incubations at 37° C. for 1 h were performed inPhA-free medium for cells treated only with PhA or in PhA-free mediumcontaining 10 μM FTC for cells treated with both PhA and FTC. PhAfluorescence was measured on a FACScanto flow cytometer equipped with a635-nm red diode laser.

Functional assays of ABCB1 were performed as described above for ABCG2,with the substitution of rhodamine 123 for PhA and verapamil for FTC.

Western Blotting.

Cells were lysed in RIPA buffer (Cell Signaling). The proteinconcentration of the lysate was quantitated by the Pierce BCA ProteinAssay Kit (ThermoFisher Scientific) using bovine serum albumin as thestandard. Equal amounts of lysate were loaded and separated bySDS-polyacrylamide gels (4-20%), and transferred onto nitrocellulosemembranes. The membranes were blocked with 5% non-fat milk powder in TBSfor 1 h and probed with appropriate primary antibodies. After washingwith TBS-T, the membrane was incubated with anti-rabbit IgG, HRP-linkedantibody (Cell Signaling) and visualized using enhancedchemiluminescence.

In Vitro cytotoxicity. Sensitivity to SN-38 or IMMU-132 was determinedby an MTS-based assay using CELL TITER 96® AQ_(ueous) One Solution(Promega). Briefly, cells were placed into wells of 96-well plates.Working solutions of IMMU-132 (at a concentration of 2,500 nM in SN-38equivalents) and SN-38 at 2,500 nM were prepared from respective stocksolutions in sterile media. From these working solutions, serial 5-folddilutions were made in sterile media to yield final concentrationsbetween 500 and 0.0064 nM in test wells. Plates were incubated at 37° C.in a humidified chamber with 5% CO₂ for 96 h, after which 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumwas added and returned to the incubator until cells of the untreatedcontrol had an absorbance greater than 1.0. Cytotoxicity was measured asa percent of growth relative to untreated cells. Dose-response curveswere generated from the mean of triplicate determinations, and IC₅₀values were calculated using GraphPad Prism Software V 6.05 (AdvancedGraphics Software).

The effect of ABCG2 inhibitors on the IC₅₀ values of SN-38 for theparental and resistant cell lines was determined from the respectivedose-response curves obtained for SN-38 in the presence of a non-toxicconcentration of ABCG2 inhibitors.

Estimation of Double-Strand Breaks with Measurements of γH2AX-StainedCells.

Cells (5×10⁵ cells/mL per sample) were incubated with or without SN-38(250 nM) at 37° C. for the indicated times, fixed in 4% formalin for 15min, then washed and permeabilized in 0.15% TRITON™ X-100 in PBS for 15min. After washing twice with 1% bovine serum albumin-PBS, the cellswere incubated with murine anti-γH2AX-AF488 for 45 min at 4° C. Thesignal intensity of γH2AX was measured by flow cytometry using aFACSCALIBUR™ (BD Biosciences).

Reverse Transcription-Quantitative Real-Time PCR (RT-qPCR).

Total RNA was extracted using RNEASY® Mini Kit (Qiagen) andreverse-transcribed to complementary DNA using SUPERSCRIPT® IVFirst-Strand Synthesis System (Life Technologies). Quantitativereal-time PCR (qPCR) was performed for each sample in triplicate on aBioRad CFX96 Real-Time System using the TAQMAN® Gene Expression Assay(with primer sets of Hs01053790 ml for ABCG2 and Hs99999905 ml forGAPDH) and TaqMan® Universal Master Mix II, all procured from LifeTechnologies. The mRNA level of ABCG2 was normalized to that of GAPDHand expressed as 2^(−ΔCt), where ΔCt=[Ct (ABCG2)−Ct (GAPDH)], and Ct(threshold cycle) is the number of PCR cycle corresponding to theintersection between an amplification curve and a threshold linedetermined for a target gene.

In Vivo Therapy Studies.

NCr female athymic nude (nu/nu) mice, 4 weeks old, were purchased fromTaconic Farms. NCI-N87 and NCI-N87-S120 tumor xenografts wereestablished by harvesting cells from tissue culture, making a 1:1 cellsuspension in MATRIGEL® (BD Bioscience), and injecting each mouse with atotal of 1×10⁷ cells s.c. in the right flank. Tumor volume (TV) wasdetermined by measurements in two dimensions using calipers, withvolumes defined as: L×w²/2, where L is the longest dimension of thetumor and w the shortest. Mice were randomized into treatment groups of9-10 and therapy begun when tumor volumes were approximately 0.25 cm³.Mice bearing NCI-N87-S-120 tumors were treated with irinotecan (40 mg/kgi.v., q2dx5d) or IMMU-132 (0.5 mg i.v. twice weekly for four weeks).YHO-13351 (0.6 mg i.v.) was administered at the same time the therapystarted and again at 4 h post-therapy. For the IMMU-132+ YHO-13351combination group, a third injection of YHO-13351 was administered 24 hpost-IMMU-132 administration. Control mice received each agent alone.For YHO-13351 control mice, they were injected on the same schedule aswhen combined with irinotecan. Another group of mice bearing parentalNCI-N87 tumors served as a control for efficacy of IMMU-132 andirinotecan in tumors lacking the ABCG2 pump. The lyophilized IMMU-132was reconstituted and diluted in sterile saline as required.Irinotecan-HCl injection was diluted in sterile saline and the finaldose based on body weight (40 mg/kg). Mice were euthanized and deemed tohave succumbed to disease once tumors grew to >1.0 cm³ in size.

Statistics. Statistical analysis for the tumor growth data was based onarea under the curve (AUC) and survival time. Profiles of individualtumor growth were obtained through linear curve modeling. An f-test wasemployed to determine equality of variance between groups prior tostatistical analysis of growth curves. A two-tailed t-test was used toassess statistical significance between groups. As a consequence ofincompleteness of some of the growth curves due to deaths, statisticalcomparisons of AUC were only be performed up to the time at which thefirst animal within a group was sacrificed. Log-rank analysis to comparethe Kaplan-Meier survival curves of two groups was performed withGraphPad Prism V6.05. Significance was set at P≤0.05.

Results

Establishment of SN-38-Resistant Cell Lines.

Table 14 summarizes the IC₅₀ values of the parental cell lines(MDA-MB-231 and NCI-N87) and their SN-38-resistant counterparts(MDA-MB-231-5120 and NCI-N87-S120), as determined for SN-38, doxorubicinand paclitaxel. Compared to the parental cells, both S-120 cells wereabout 50-fold more resistant to SN-38, and relatively notcross-resistant to either doxorubicin or paclitaxel. NCI-N87-S120 cellscultured for three weeks or longer in SN-38-free medium graduallyrestored their sensitivity to SN-38, resulting in a revertant cell line(NCI-N87-S120-REV) with reduced resistance to SN-38 (IC₅₀=50 nM) over aperiod of 4 months.

TABLE 14 Sensitivity of the parental and SN-38-resistant cell lines toselected drugs. IC₅₀ (nM) NCI-N87- MDA-MB- MDA-MB- Drug NCI-N87 S120RF^(a) 231 231-S120 RF^(a) SN-38 4.3 ± 0.7 211 ± 39  49.1  4.8 ± 1.7 248± 79 51.7  SN-38 + YHO- 3.9 ± 0.5 6.4 ± 1.6 1.6 2.4  16 ± 11 6.713351^(b) SN-38 + Ko143^(b) ND 1.3 ± 0.4 ND ND ND ND Doxorubicin 32.2 ±7.5  74.6 ± 3.3  2.3 10.9 ± 0.6  43.1 ± 4.6 4.0 Paclitaxel 7.9 ± 4.0 8.6± 6.5 1.1 2.5 ± 0.1  3.2 ± 0.7 1.3 ^(a)RF is the resistance factorobtained as the [IC₅₀ mean of S120] divided by the [IC₅₀ mean ofparent]. ^(b)The non-toxic concentrations used for YHO-13351 and Ko143were 2 and 1 μM, respectively.

Overexpression of Functional ABCG2 in the S-120 Cell Lines.

The presence of ABCG2 in the S-120 cells, but little, if any, in theparental cells was shown by Western blot analysis (data not shown) andcorroborated by RT-qPCR, which indicated that virtually no mRNAtranscripts of ABCG2 could be detected in MDA-MB-231 and NCI-N87 cells(data not shown). On the other hand, the mRNA levels of ABCG2 relativeto GAPDH in MDA-MB-231-S120 and NCI-N87-S120 are calculated to be27,408-fold and 167-fold higher than those in MDA-MB-231 and NCI-N87,respectively (data not shown). The expression of ABCG2 was alsoconfirmed in samples obtained from NCI-N87-S120, but not NCI-N87,xenografts (data not shown).

That ABCG2 is functionally active in the two S-120 cell populations, butabsent in the parental cells, was demonstrated by histogram analysis forPhA, which is a fluorescent substrate of ABCG2. In both parental cells,the intracellular levels of PhA, as measured by the median fluorescenceintensity (MFI), were relatively high and remained practically the samewith or without FTC (data not shown), a potent mycotoxin initiallyidentified for its effective reversal of resistance to mitoxantrone,doxorubicin, and topotecan in a multidrug-selected cell line. Bycontrast, in either S-120 cells, a high level of intracellular PhAsimilar to that in the parental cells could only be observed with theaddition of FTC, whereas the omission of FTC resulted in a greater than95% reduction of intracellular PhA (data not shown). Similar resultswere obtained in human lung cancer A549 cells, known to expressfunctional ABCG2 (Scharenberg et al., 2002, Blood 99:507-12). Inseparate studies with rhodamine 123 and verapamil as the substrate andthe inhibitor for ABCB1, respectively, activity of ABCB1 was detected inhuman colorectal cancer HCT15 cells, which served as a positive controlfor expressing ABCB1 (Alvarez et al., 1995, J Clin Invest 1995;95:2205-14), but not in either NCI-N87-S120 or NCI-N87 (data not shown).

The activity of ABCG2 in the two S-120-resistant cell lines also wasdemonstrated by comparing the levels of DNA double-strand breaks (DSBs)induced by SN-38 with those in the parental cells, as measured by a flowcytometric assay for quantification of γH2AX, whose signal intensitiesdirectly correspond to the number of DSBs formed (Rogakou et al., 1998,J Biol Chem 273:5858-68). As shown in FIG. 36A, upon treatment with 250nM of SN-38 the levels of γH2AX rose steadily in the parental, but notthe resistant S-120, cells, culminating, after 3 h, in an increase overthe untreated controls that was about 2-fold for MDA-MB-231 and about4-fold for NCI-N87 (FIG. 36B). ABCG2 is implicated in preventing theincrease of γH2AX in the S-120 cells treated with SN-38, since theaddition of both FTC (10 μM) and SN-38 (250 nM) to MDA-MB-231-5120 couldelevate γH2AX levels with a comparable fold-increase to that ofSN-38-treated MDA-MB-231 (FIG. 36C). Similar results were obtained withNCI-N87-S120 when treated with SN-38 or IMMU-132 in the presence of FTC(FIG. 36D).

Sensitizing S-120 Cells to SN-38 with Selected ABCG2 Inhibitors.

The effect of two known ABCG2 inhibitors, Ko143 (Allen et al., 2002, MolCancer Ther 1:417-25) and YHO-13351 (Yamazaki et al., 2011, Mol CancerTher 10:1252-63), on reversing the resistance of S-120 cells to SN-38was examined in vitro at a concentration not affecting the growth ofeither the parental or the S-120 cells. As shown in FIG. 37A-C of therepresentative dose-response curves, and in Table 14 of thecorresponding IC₅₀ values, the addition of either ABCG2 inhibitor, whileconferring little impact on the sensitivity of parental cells to SN-38,reduced the IC₅₀ of SN-38 by more than 90% in both resistant S-120 celllines. Limited studies also were done for other ABCG2 inhibitors, suchas FTC (Rabindran et al., 2000, Cancer Res 60:47-50), cyclosporine A(Qadir et al., 2005, Clin Cancer Res 11:2320-6), and GF120918 (de Bruinet al., 1999, Cancer Lett 146:117-26), with results showing comparableor less potency (data not shown). Noting the reported instability ofKo143 in rat serum (Weidner et al., 2015, 354:384-93), YHO-13351 wasselected over Ko143 for in vivo evaluation.

Improved efficacy of IMMU-132 when combined with YHO-13351 inSN-38-resistant NCI-N87-S-120 tumors. NCI-N87-S-120 tumors grew moreslowly in the mice than did parental NCI-N87 (FIG. 38A; P=0.005, AUC),with the median survival for untreated animals more than 2-fold longerfor the mice bearing NCI-N87-S-120 (P=0.0006 vs. NCI-N87). Whiletreatment with IMMU-132 or irinotecan provided no significant survivalbenefit to mice bearing NCI-N87-S-120 tumors (FIG. 38B), both of thesetherapies resulted in a greater than 2-fold increase in survival in micebearing NCI-N87 (P<0.0001; FIG. 38C). However, when IMMU-132 therapy wascombined with YHO-13351 in mice bearing SN-38-resistant NCI-N87-S-120, asignificant 64% improvement in survival was achieved in comparison tountreated animals (P=0.0278). Although irinotecan plus YHO-13551likewise improved the survival of the mice, it did not reachsignificance (P=0.0852). Since the in vitro assay showed there was nodifference between parental cell lines treated with SN-38 alone or incombination with YHO-13351, and the tumor xenograft samples of NCI-N87were absent of ABCG2 by Western blot (not shown), a combination ofIMMU-132 and YHO-13351 was not examined in mice bearing NCI-N87.

DISCUSSION

IMMU-132 is a first-in-class ADC made by conjugating the moderatelytoxic drug, SN-38 (nanomolar potency), with a partially stable linker,site-specifically and at a high DAR of 7.6, to a humanized antibodyagainst Trop-2, an antigen expressed in many solid cancers. Preclinicalstudies have demonstrated that IMMU-132, in comparison to irinotecan,protects the IgG-bound SN-38 from glucuronidation and delivers much moreSN-38 (20- to 136-fold higher) to tumor xenografts, resulting inimproved pharmacokinetics and pharmacodynamics (Sharkey et al., 2015,Clin Cancer Res 21:5131-8). As such, IMMU-132 provides a paradigm changethat contrasts the prevailing approach of conjugating a low level of anultratoxic payload (picomolar potency) with a stable linker to anantibody capable of internalization upon target engagement (Sievers etal., 2013, Annu Rev Med 64:15-29; Gordon et al., 2015, Bioconjugate Chem26:2198-2215).

Importantly, the ongoing Phase II studies with IMMU-132 as a singleagent in patients with metastatic triple-negative breast cancer (mTNBC)who had received a median of 5 (range=2 to 12) prior lines of therapyhave shown an interim objective response rate of 31% by RECIST1.1 in 58evaluable patients (Bardia et al., Safety and efficacy of anti-Trop-2antibody drug conjugate, sacituzumab govitecan (IMMU-132), in heavilypretreated patients with TNBC. San Antonio Breast Cancer Symposium2015), thus extending the results obtained in Phase I trials whichindicated IMMU-132 had acceptable toxicity and encouraging therapeuticactivity in patients with difficult-to-treat solid cancers (Starodub etal., 2015, Clin Cancer Res 21:3870-8). Promising initial results alsohave been reported for IMMU-132 administered to patients withplatinum-resistant urothelial carcinoma (Faltas et al., 2016, ClinGenitourinary Cancer 14:e75-9). Whereas the Phase II results observed inheavily pretreated patients with mTNBC have led the FDA to grantBreakthrough Therapy Designation to IMMU-132, those who showed earlyprogression of disease, thus failing IMMU-132, may reflect possible drugresistance resulting from one or more preexisting efflux pumps, sinceSN-38 is susceptible to multiple ABC transporters (Szakacs et al., 2006,Nat Rev Drug Discov 5:219-34).

In the current Example, NCI-N87-S120 and MDA-MB-231-5120, the twosublines made resistant to SN-38, were shown to be 50-fold lessresponsive to SN-38 than their parental cells. The sensitivity ofNCI-N87-S120 to SN-38 could be restored to within 5-fold of NCI-N87 whenpropagated in vitro without SN-38 after a period of 3 weeks or longer,suggesting a non-genetic origin of such acquired resistance. Thepresence of ABCG2 in the two S120 sublines, but not their parents, wassupported by several lines of evidence, including the demonstration ofactive efflux of PhA; the detection of expressed protein by Westernblotting, which was corroborated with RT-qPCR of mRNA; the increasedaccumulation of SN-38 by FTC using γH2AX as a surrogate marker; and thereverted resistance by YHO-13351 or Ko143. Of note, both S-120 sublineswere found not to carry ABCB1 and neither phenotype was cross-resistantto doxorubicin or paclitaxel, similar to a previous observation (Candeilet al., 2004, Int J Cancer 2004; 109:848-54) that the ABCG2-expressing,SN-38-resistant human colorectal HCT116-SN50 cancer subline showed nosignificant cross-resistance to doxorubicin (as well as to5-fluorouracil and oxaliplatin). In other human cancer clones selectedfor resistance to SN-38 via continuous exposure of parental cell linesto the drug in culture, over-expression of ABCG2 also has been reportedfor the sublines generated from MCF-7 (Imai et al., 2014, JSM Clin OncolRes 2:1008; Jandu et al., 2016, BMC Cancer 16:34), MDA-MB-231 (Jandu etal., 2016, BMC Cancer 16:34), the small-cell lung carcinoma PC-6(Kawabata et al., 2001, Biochem Biophys Res Commun 280:1216-23), thenon-small-cell lung cancer adenocarcinoma H23 (Bessho et al., 2006,Cancer Sci 97:192-8), and the cervical cancer carcinoma HeLa (Takara etal., 2009, Cancer Lett 278:88-96). Cross-resistance of these sublines todoxorubicin varied somewhat, with the resistance ratio (IC₅₀ inresistant subline divided by IC₅₀ in the parental cell) being less than1.3 for the sublines of PC-6 (Kawabata et al., 2001, Biochem Biophys ResCommun 280:1216-23), 2.5 for the sublines of HeLa (Takara et al., 2009,Cancer Lett 278:88-96), and about 7.0 for the subline of H23 (Bessho etal., 2006, Cancer Sci 97:192-8). Whereas cross-resistance to doxorubicinwas not determined for the SN-38 resistant sublines derived from MCF-7(Imai et al., 2014, JSM Clin Oncol Res 2:1008; Jandu et al., 2016, BMCCancer 16:34) or MDA-MB-231 (Jandu et al., 2016, BMC Cancer 16:34),these sublines remained sensitive to vincristine (Imai et al., 2014, JSMClin Oncol Res 2:1008), cisplatin (Imai et al., 2014, JSM Clin Oncol Res2:1008; Jandu et al., 2016, BMC Cancer 16:34) and docetaxel (Jandu etal., 2016, BMC Cancer 16:34).

When cultured in vitro, SN-38-resistant sublines of MCF-7 or MDA-MB-231had longer doubling times than their parental cells (Jandu et al., 2016,BMC Cancer 16:34). Thus, it is not surprising that NCI-N87-S120xenografts grew significantly slower in the mice than NCI-N87, which isconsistent with the notion that drug-resistant tumor sublines selectedin vitro frequently manifest less aggressive properties than theirdrug-sensitive parental cell lines (Kerbel et al., 1994, J Cell Biochem56:37-47). Nevertheless, in vivo studies show that the NCI-N87-S-120xenograft retained ABCG2 expression and was resistant to IMMU-132, yetits growth could be significantly subdued by IMMU-132 in combinationwith YHO-13351. A parallel study shows the parental xenograft wasresponsive to IMMU-132 or irinotecan, but with a shorter median survivaltime. Together, these in vivo results suggest that suitable inhibitorsthat are tolerated well by the host animals can overcome ADC resistanceand that the resistant tumor lines can become appreciably responsive toIMMU-132 and to a lesser extent to irinotecan. It further appears thattesting for drug resistance is of use as a predictive bioassay (Jandu etal., 2016, BMC Cancer 16:34, Bessho et al., 2006, Cancer Sci 97:192-8)to select patients who should receive ABC-blocking therapy withIMMU-132. It further appears that clinically-tested tyrosine kinaseinhibitors, some of which interfere with the functions of ABCtransporters at non-toxic levels (Anreddy et al., 2014, Molecules19:13848-77), are of use to enhance the potency of IMMU-132 in cancercells that are intrinsically or made resistant to SN-38.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usage andconditions without undue experimentation. All patents, patentapplications and publications cited herein are incorporated byreference.

We claim:
 1. A method of treating a subject with a Trop-2 positivecancer comprising: a. administering to the subject an anti-Trop-2antibody-drug conjugate (ADC) comprising SN-38 conjugated to ananti-Trop-2 hRS7 antibody; and b. administering to the subject an ABCG2inhibitor selected from the group consisting of fumitremorgin C, Ko143,GF120918 and YHO-13351; wherein the subject is resistant to or relapsedfrom therapy with a checkpoint inhibitor.
 2. The method of claim 1,wherein the ABCG2 inhibitor is YHO-13351.
 3. The method of claim 1,wherein the ADC is administered at a dosage of between 8 mg/kg and 10mg/kg.
 4. The method of claim 1, wherein the subject is resistant to orrelapsed from therapy with irinotecan, topetecan or SN-38.
 5. The methodof claim 1, further comprising: (i) screening cancer cells from thesubject for sensitivity to the ADC; and (ii) selecting subjects fortherapy with the ADC and ABCG2 inhibitor whose cancer cells areresistant to therapy with the ADC alone.
 6. The method of claim 1,wherein the cancer is selected from the group consisting of colorectal,lung, stomach, urinary bladder, renal, pancreatic, breast, ovarian,uterine, esophageal and prostatic cancer.
 7. The method of claim 1,wherein the treatment results in a reduction in tumor size of at least15%, at least 20%, at least 30%, or at least 40%.
 8. The method of claim1, wherein the cancer is selected from the group consisting oftriple-negative breast cancer, HER+, ER+, progesterone+ breast cancer,metastatic non-small-cell lung cancer, metastatic small-cell lungcancer, metastatic urothelial cancer and metastatic pancreatic cancer.9. The method of claim 1, wherein the ADC comprises 6 to 8 molecules ofSN-38 conjugated to the antibody or antigen-binding fragment thereof.10. The method of claim 1, wherein the cancer is metastatic.
 11. Themethod of claim 10, further comprising reducing in size or eliminatingthe metastases.
 12. The method of claim 1, wherein there is a linkerbetween the SN-38 and the antibody.
 13. The method of claim 12, whereinthe linker is CL2A and the structure of the ADC is MAb-CL2A-SN-38


14. The method of claim 13, wherein the 10-hydroxy position of SN-38 inMAb-CL2A-SN-38 is a 10-O-ester or 10-O-carbonate derivative using a‘COR’ moiety, wherein “CO” is carbonyl and the “R” group is selectedfrom (i) an N,N-disubstituted aminoalkyl group “N(CH₃)₂—(CH₂)_(n)—”wherein n is 1-10 and wherein the terminal amino group is optionally inthe form of a quaternary salt; (ii) an alkyl residue “CH₃—(CH₂)_(n)—”wherein n is 0-10; (iii) an alkoxy moiety “CH₃—(CH₂)_(n)—O—” wherein nis 0-10; (iv) an “N(CH₃)₂—(CH₂)_(n)—O—” wherein n is 2-10; or (v) an“R₁O—(CH₂—CH₂—O)_(n)—CH₂—CH₂—O—” wherein R₁ is ethyl or methyl and n isan integer with values of 0-10.
 15. The method of claim 1, wherein theantibody comprises the light chain CDR sequences CDR1 (KASQDVSIAVA, SEQID NO: 14); CDR2 (SASYRYT, SEQ ID NO: 15); and CDR3 (QQHYITPLT, SEQ IDNO: 16) and the heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO: 17);CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO: 18) and CDR3 (GGFGSSYWYFDV, SEQ IDNO: 19).
 16. The method of claim 1, further comprising administering tothe subject at least one other anti-cancer therapy selected from thegroup consisting of surgery, external radiation, radioimmunotherapy,immunotherapy, chemotherapy, antisense therapy, interference RNAtherapy, treatment with a therapeutic agent and gene therapy.
 17. Themethod of claim 16, wherein the therapeutic agent is a drug, toxin,immunomodulator, second antibody, antigen-binding fragment of a secondantibody, pro-apoptotic agent, toxin, RNase, hormone, radionuclide,anti-angiogenic agent, siRNA, RNAi, chemotherapeutic agent, cytokine,chemokine, prodrug or enzyme.
 18. The method of claim 17, wherein thedrug is selected from the group consisting of 5-fluorouracil, afatinib,aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101,AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1,busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatin,Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine,camptothecans, crizotinib, cyclophosphamide, cytarabine, dacarbazine,dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubicin,doxorubicin, cyano-morpholino doxorubicin, doxorubicin glucuronide,epirubicin glucuronide, erlotinib, estramustine, epipodophyllotoxin,erlotinib, entinostat, estrogen receptor binding agents, etoposide(VP16), etoposide glucuronide, etoposide phosphate, exemestane,fingolimod, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO),fludarabine, flutamide, farnesyl-protein transferase inhibitors,flavopiridol, fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib,gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide,imatinib, L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13,lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine,vinblastine, vincristine, vinca alkaloids and ZD1839.
 19. The method ofclaim 17, wherein the immunomodulator is selected from the groupconsisting of cytokines, lymphokines, monokines, stem cell growthfactors, lymphotoxins, hematopoietic factors, colony stimulating factors(CSF), interferons (IFN), parathyroid hormone, thyroxine, insulin,proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepaticgrowth factor, prostaglandin, fibroblast growth factor, prolactin,placental lactogen, OB protein, transforming growth factor (TGF), TGF-α,TGF-β, insulin-like growth factor (IGF), erythropoietin, thrombopoietin,tumor necrosis factor (TNF), TNF-α, TNF-β, mullerian-inhibitingsubstance, mouse gonadotropin-associated peptide, inhibin, activin,vascular endothelial growth factor, integrin, interleukin (IL),granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-β, interferon-γ, S1 factor, IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18 IL-21, IL-23, IL-25, LIF, kit-ligand, FLT-3, angiostatin,thrombospondin and endostatin.
 20. The method of claim 1, furthercomprising administering to the subject an inhibitor of ABCB1 or ABCC1.21. The method of claim 1, further comprising administering to thesubject a tyrosine kinase inhibitor.